scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

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

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1683-1687 ◽  
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.

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

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1683-1687 ◽  
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

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.

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

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 924-928 ◽  
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.

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

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 299-304 ◽  
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.

scholarly journals The in vitro synthesis of polypeptides for the platelet membrane glycoproteins IIb and IIIa

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1031-1037 ◽  
Author(s):  
SM Silver ◽  
MM McDonough ◽  
G Vilaire ◽  
JS Bennett
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Platelet Membrane ◽  
Human Leukemia ◽  
Membrane Glycoproteins ◽  
In Vitro Synthesis ◽  
Calcium Dependent ◽  
Hel Cells ◽  
Von Willebrand

Abstract The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium- dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.

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

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 354-359 ◽  
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.

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

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 924-928 ◽  
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.

scholarly journals 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 ◽  
2008 ◽  
Vol 112 (2) ◽  
pp. 340-349 ◽  
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.

scholarly journals Adamts13-Cultured Red Blood Cells to Treat Thrombotic Thrombocytopenic Purpura

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 89-89
Author(s):  
Kai Wang ◽  
Khulan Batbayar ◽  
Karl Roberts ◽  
Emmanuel Olivier ◽  
Eric E. Bouhassira
Keyword(s):  
Red Blood Cells ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Blood Cells ◽  
Vascular System ◽  
Conflicts Of Interest ◽  
Direct Injection ◽  
Culture Method ◽  
Thrombocytopenic Purpura ◽  
Membrane Bound

Red Blood Cells (RBCs) have long been considered a potentially useful means of delivering drugs to the circulation because delivery through therapeutic RBCs as compared to direct injection in the plasma can lengthen the half-life of the therapeutic agent in the circulation, spatially restrict the drugs to the lumen of the cardio-vascular system, and shield the drug from the immune system. Despite some progress, loading the cells with therapeutically useful cargo remains technically challenging. We have recently developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into unlimited numbers of enucleated cultured RBCs. This provides an ideal method to produce therapeutic RBCs since iPSCs can be genetically manipulated with powerful CRISPR-based technologies. ADAMTS13, whose deficiency is responsible for congenital and acquire Thrombotic Thrombocytopenic Purpura (TTP) is a good target as a therapeutic that could be delivered through drug-carrying RBCs because large amounts of plasma concentrate, or more recently recombinant proteins, are necessary to treat TTP. We report here we have produced engineered erythroid cells that contains globin-LCR driven ADAMTS13 fusion transgenes inserted at safe harbor AAVS1, and that these cells express a membrane bound version of an inhibitor-resistant version of ADAMTS13. We show using flow cytometry that the fusion protein is express at high levels, and using a FRET assay that detect cleavage of the von Willebrand cognate site, that the membrane bound ADAMTS13 is enzymatically active. Comparison of enzymatic activity with plasma concentrate suggests that about 50 billion engineered ADAMTS13-cRBCs would be sufficient to deliver an amount of ADAMTS13 equivalent to 2 liters of plasma concentrate. This suggests that a transfusion of about 10 mL of ADAMTS13-RBCs could be therapeutic for congenital and acquired TTP. The number of cRBCs necessary to treat even a few patients is very large. This has been considered a major obstacle to the development of treatment based on in vitro produced RBCs because of the volume of culture that is necessary to produce the cells. We also report that we have developed a culture method based on holo-fiber bioreactors that allows the production of cRBCs at a density of 5.108 cell/mL which is sufficient to produce enough cells to performed small clinical trials. Disclosures No relevant conflicts of interest to declare.

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