ADAMTS13 Autoantibodies, Antigen and Proteolytic Activity in Patients with Thrombotic Thrombocytopenic Purpura.

Abstract Many patients with thrombotic thrombocytopenic purpura (TTP) harbor autoantibodies against the ADAMTS13 protease, which can block proteolytic activity or accelerate clearance of this protease. The autoantibodies may be categorized as ‘inhibitory’ antibodies that bind and inhibit ADAMTS13 activity in vitro and ‘non-inhibitory’ antibodies that may bind, but do not affect the ADAMTS13 activity. The pathophysiologic role of two types of anti-ADAMTS13 autoantibodies on the level of ADAMTS13 antigen and activity remains unclear. To address this question, the relationship between the autoantibodies, and the levels of antigen and proteolytic activity in 40 patients with TTP (26 idiopathic and 14 non-idiopathic) was determined. The diagnosis of TTP was based on the presence of thrombocytopenia and microangiopathic hemolytic anemia with or without renal dysfunction and neurological symptoms. The ADAMTS13 antigen was determined by ELISA (American Diagnostica, Stamford CT). The ADAMTS13 autoantibodies were determined by Technozyme-ELISA (Technoclone GmbH, Vienna, Austria) and Immunoprecipitation and Western blotting. The ADAMTS13 activity was determined by Fluorescent resonance energy transfer (FRETS)-VWF73. The results showed that: 1) autoantibodies were present in 24 of 40 (60%) of all TTP patients, 19 of 26 (73%) in idiopathic TTP and 5 of 14 (36%) in non-idiopathic TTP. The prevalence of non-inhibitory autoantibodies was approximately 31% (8/26) in patients with idiopathic TTP and 36% (5/14) in patients with non-idiopathic TTP; 2) there was a positive correlation between the ADAMTS13 antigen levels and enzymatic activity in 36 samples tested (r=0.665, P<0.0001). This positive correlation was not altered even after removing from the analysis samples demonstrating inhibitors and low activity; 3) with respect to antigen levels, there was no significant difference between idiopathic vs. non-idiopathic TTP patients, whereas ADAMTS13 activity are significantly lower in the former (Table 1); 4) the inhibitory autoantibodies significantly reduce ADAMTS13 activity and may or may not decrease ADAMTS13 antigen levels. However, non-inhibitory autoantibodies neither decrease ADAMTS13 levels nor proteolytic activity (Table 2). We conclude that overall, in TTP patient samples, ADAMTS13 antigen levels correlate with activity. Subgroup analysis indicates that idiopathic and non-idiopathic TTP patients have similar ADAMTS13 antigen levels, whereas the former demonstrates much less proteolytic activity. TTP patients with inhibitory autoantibodies may have a more profound effect on ADAMTS13 activity (and perhaps also on clearance) than those with non-inhibitory autoantibodies. The non-inhibitory antibodies do not appear to accelerate ADAMTS13 protease clearance in vivo. Table 1. ADAMTS13 Antigen and Activity in Idiopathic and Non-Idiopathic TTP Patients Patients Antigen (ng/ml) Activity (%) * p < 0.05 Idiopathic 276 ± 46 (n=19) 16.9 ± 6 (n=21)* Non-idiopathic 328 ± 43 (n=17) 45.1 ± 5.7 (n=19) Table 2. Effect of Autoantibodies on ADAMTS13 Antigen and Activity Antibodies Definition Antigen (ng/ml) Activity (%) *p=0.085; **p < 0.05 vs "non-inhibitory" and <0.05 vs. "None" group Inhibitory Activity <10%, Inhibitor (1:1), positive, TecZym>15 U/ml 183 ± 55* 4.1 ± 1.2 (n=9)** Non-inhibitory Activity>10%, Inhibitor (1:1), negative, TecZym >15 U/ml 368 ± 52 37.6 ± 8 (n=17) None Activity >10%, Inhibitor (1:1), negative, TecZym <12 U/ml 294 ± 51 40.2 ± 7 (n=13)

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The Frequency and the Significance of ADAMTS-13 Neutralising Inhibitors in 62 Patients with Non-Familial Thrombotic Thrombocytopenic Purpura.

Blood ◽

10.1182/blood.v104.11.3945.3945 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3945-3945

Author(s):

Martina Böhm ◽

Manuela Krause ◽

Charis Von Auer ◽

Wolfgang Miesbach ◽

Inge Scharrer

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Inhibitory Activity ◽

Thrombocytopenic Purpura ◽

Plasma Exchange Therapy ◽

Normal Plasma ◽

Initiation Of Therapy ◽

Inhibitor Titer ◽

Ristocetin Cofactor

Abstract Neutralising inhibitors against ADAMTS-13 are detected in 51–67% of patients with Thrombotic Thrombocytopenic Purpura (TTP). These ADAMTS-13 inhibitors have not been very well characterised and the diagnostic or the prognostic value of these inhibitors is not established. In the present study, we measured ADAMTS-13 activity and the corresponding inhibitor titer in 96 samples from 62 patients with TTP at various stages of their disease. All patients presented with non-familial TTP. For patients with severe ADAMTS-13 activity without detectable inhibitor heritable ADAMTS-13 deficiency was excluded by either normalisation of ADAMTS-13 activity in remission or by detecting normal ADAMTS-13 activity in first degree family members. ADAMTS-13 activity was quantified by measuring the residual ristocetin cofactor activity of the substrate. The inhibitor against ADAMTS-13 was detected by mixing patient plasma, either neat or diluted, with normal plasma. The inhibitor concentration neutralising 50% of ADAMTS-13 activity in a 1:1 dilution of patient plasma with normal plasma was defined as 1 U/ml. Inhibitors were considered non-detectable (<0.4 U/ml), if residual ADAMTS-13 activity in the mixture was higher than 75%. Samples with ADAMTS-13 activity >6.25% were heat-inactivated (30 min at 56°C) before testing for inhibitory activity. We found severe ADAMTS-13 deficiency in 89% (24/27) of the samples from patients with acute untreated TTP. 87% (21/24) of these samples were positive for inhibitory activity. The inhibitor titer ranged from 0.4 to 62 U/ml with a median of 1 U/ml. One patient with acute TTP demonstrated ADAMTS-13 activity of 34% despite an inhibitor titer of 0.6 U/ml. The sensitivity of a positive inhibitor test for the diagnosis of TTP was thus 82%. The inhibitor titer before initiation of therapy could not be correlated with the platelet count, the CRP-level or the response to PE-therapy, if patients with an index episode and patients with a relapse were analysed separately. Severe ADAMTS-13 activity was detected in 15/31 samples collected from patients during plasma exchange therapy. 93% (14/15) of these samples were positive for an inhibitor with a titer ranging between 0.6 and 47 U/ml (median: 3 U/ml). 12 of 37 patients tested in remission presented with severe ADAMTS-13 deficiency, 6 of them were positive for inhibitory activity (range: 1–52 U/ml; median: 5 U/ml). The inhibitor titer for patients, which were analysed during acute untreated TTP as well as in remission (n=5), was notable not related to the stage of disease. Five patients with positive inhibitory activity at admission demonstrated mild ADAMTS-13 deficiency in remission without detectable inhibitor. In contrast, we detected inhibitory activity of 0,6–0,8 U/ml in 3 samples from two patients with measurable ADAMTS-13 activity. These low titer inhibitors were only detectable, if samples were heat inactivated before performing the inhibitor assay. Our data demonstrate, that inhibitors against ADAMTS-13 are very heterogeneous. It is highly suspected, that some of these inhibitors can either completely or partly neutralise ADAMTS-13 function in vivo without being detectable in vitro. Inhibitors against ADAMTS-13 might, on the other side, not always completely inhibit ADAMTS-13 function, since they can occur in patients with high residual ADAMTS-13 activity. Inhibitor titers show a wide variation and the clinical significance of the inhibitor titer before, during and after therapy needs to be further investigated.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.bloodjournal693924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 1

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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Platelet-agglutinating protein P37 from a thrombotic thrombocytopenic purpura plasma forms a complex with human immunoglobulin G

Blood ◽

10.1182/blood.v71.2.299.299 ◽

1988 ◽

Vol 71 (2) ◽

pp. 299-304 ◽

Cited By ~ 19

Author(s):

FA Siddiqui ◽

EC Lian

Keyword(s):

Complex Formation ◽

Thrombotic Thrombocytopenic Purpura ◽

Fluid Phase ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombocytopenic Purpura ◽

Form Complex ◽

Specific Igg ◽

Sepharose 4B

Abstract We have previously reported the purification of a 37-kd platelet- agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been shown to be inhibited by IgG from normal human adults and the same TTP patient after recovery. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG from normal adults and the same TTP patient after recovery to adsorbed PAP by using an enzyme-linked immunosorbent assay. The binding was specific, concentration dependent, and saturable. IgG purified from a 5-month-old baby and the same TTP patient during active disease did not form complex with PAP p37. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP p37 but not 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP p37 and IgG also revealed the fluid-phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The IgG that was bound to PAP p37 conjugated to Sepharose 4B inhibited the agglutination of washed platelets induced by TTP plasma containing PAP p37, whereas the IgG that was not bound to PAP p37 did not have a significant inhibitory effect. The complex formation between PAP p37 and specific IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and, at least partly, the in vivo successful treatment with specific IgG-containing normal plasma.

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Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v70.5.1683.1683 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1683-1687 ◽

Cited By ~ 42

Author(s):

WG Murphy ◽

JC Moore ◽

JG Kelton

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Cysteine Protease ◽

Platelet Activating Factor ◽

Aminocaproic Acid ◽

Human Platelets ◽

Thrombocytopenic Purpura ◽

Platelet Surface ◽

Calcium Dependent

Abstract Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1- arginine chloromethyl ketone. These studies suggested that the platelet- activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.

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Effects of platelet inhibitors on the platelet aggregation induced by plasma from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v58.2.354.354 ◽

1981 ◽

Vol 58 (2) ◽

pp. 354-359 ◽

Cited By ~ 23

Author(s):

EC Lian ◽

N Savaraj

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Creatine Phosphate ◽

Antiplatelet Drugs ◽

Platelet Inhibitors ◽

Prostaglandin I2 ◽

Thrombocytopenic Purpura ◽

Dextran 70

Abstract Antiplatelet drugs have been used in the treatment of thrombotic thrombocytopenic purpura (TTP) but there in vivo efficacy remains controversial. It has been shown that, in vitro, the plasmas obtained from patients with TTP induced the aggregation of washed platelets from normal donors as well as patients in remission. The effects of platelet inhibitors on the TTP plasma-induced platelet aggregation were examined. It was found that aspirin, indomethacin, ibuprofen, sulfinpyrazone, 5, 8, 11, 14-eisacotetraynoic acid, prostaglandin E1, prostaglandin I2, dBcAMP, apyrase, creatine phosphate/creatine phosphokinase, antimycin, 2-deoxy-D-glucose, dipyridamole, clofibrate, dextran 40, dextran 70, dibucaine, xylocaine, methylmaleimide, and ethylenediamine tetraacetic acid had little or no effect at all. These data lead us to conclude that at least in certain cases, antiplatelet drugs probably play a limited role in the treatment of patients with TTP.

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Studies investigating platelet aggregation and release initiated by sera from patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v69.3.924.924 ◽

1987 ◽

Vol 69 (3) ◽

pp. 924-928 ◽

Cited By ~ 35

Author(s):

JG Kelton ◽

JC Moore ◽

WG Murphy

Keyword(s):

Platelet Aggregation ◽

Thrombotic Thrombocytopenic Purpura ◽

Disease Process ◽

The Other ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Thrombocytopenic Purpura ◽

Induced Aggregation

Abstract Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

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

Blood ◽

10.1182/blood-2007-10-119552 ◽

2008 ◽

Vol 112 (2) ◽

pp. 340-349 ◽

Cited By ~ 12

Author(s):

Radu Stefanescu ◽

Dustin Bassett ◽

Rozbeh Modarresi ◽

Francisco Santiago ◽

Mohamad Fakruddin ◽

...

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Interferon Γ ◽

Microvascular Endothelial Cell ◽

Caspase 8 ◽

Inhibitory Protein ◽

Thrombocytopenic Purpura ◽

Ifn Γ ◽

Interfering Rna

Abstract 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)–γ 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-γ 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.

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FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

Thrombosis and Haemostasis ◽

10.1160/th13-08-0688 ◽

2014 ◽

Vol 112 (08) ◽

pp. 297-303 ◽

Cited By ~ 13

Author(s):

Ilaria Mancini ◽

Carla Valsecchi ◽

Luca Lotta ◽

Louis Deforche ◽

Silvia Pontiggia ◽

...

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Proteolytic Activity ◽

Binding Activity ◽

Adamts13 Activity ◽

Flow Conditions ◽

Thrombocytopenic Purpura ◽

Severe Deficiency ◽

Adamts13 Deficiency ◽

Von Willebrand ◽

Willebrand Factor

SummaryCollagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

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Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

Blood ◽

10.1182/blood.v74.3.978.bloodjournal743978 ◽

1989 ◽

Vol 74 (3) ◽

pp. 978-983 ◽

Cited By ~ 57

Author(s):

PM Mannucci ◽

R Lombardi ◽

A Lattuada ◽

P Ruggenenti ◽

GL Vigano ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Thrombotic Thrombocytopenic Purpura ◽

Von Willebrand Factor ◽

Relative Increase ◽

Thrombocytopenic Purpura ◽

Uremic Syndrome ◽

Von Willebrand ◽

Willebrand Factor

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.

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PLATELET AGGLUTINATING PROTEIN P37 FROM A THROMBOTIC THROMBOCYTOPENIC PURPURA PLASMA FORMS A COMPLEX WITH IMMUNOGLOBULIN G

10.1055/s-0038-1644589 ◽

1987 ◽

Author(s):

F A Siddigui ◽

E C -Y Lian

Keyword(s):

Complex Formation ◽

Thrombotic Thrombocytopenic Purpura ◽

Fluid Phase ◽

Sucrose Density Gradient ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombocytopenic Purpura ◽

Mechanism Of Inhibition ◽

Phase Complex

A 37-KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP), has been shown to be present in a subset of TTP patients. The platelet agglutination induced by PAP p37 has been demonstrated to be inhibited by igG from normal adults. To elucidate the mechanism of inhibition of IgG, the interaction between PAP p37 and IgG was studied. The complex formation was demonstrated by the binding of fluid-phase IgG to adsorbed PAP using enzyme-linked immunosorbent assay. The binding was specific, concentration dependent and saturable. The IgG covalently cross-linked to Sepharose 4B bound 125I-PAP but not to 125I-fibrinogen. Sucrose density gradient ultracentrifugation of a mixture of 125I-PAP and IgG also revealed the fluid phase complex formation with a sedimentation value of 19S. Complexes of molecular weight ranging from 180,000 to over 350,000 daltons were also detected by molecular sieve chromatography. The specific complex formation between PAP p37 and IgG is likely to account for the in vitro inhibition of TTP plasma-induced agglutination and , at least partly, the in vivo successful treatment with IgG-containing normal plasma.

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