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

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

Blood ◽

10.1182/blood.v70.5.1683.bloodjournal7051683 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1683-1687 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v80.9.2246.2246 ◽

1992 ◽

Vol 80 (9) ◽

pp. 2246-2251 ◽

Cited By ~ 52

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.

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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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IS IT POSSIBLE TO IDENTIFY HUMAN PLATELETS IN VITRO AS BEING DESENSITIZED TO PLATELET ACTIVATING FACTOR (PAF-ACETHER,PAF) IN VIVO?

10.1055/s-0038-1643484 ◽

1987 ◽

Author(s):

Cs Perger ◽

A von Felten

Keyword(s):

Platelet Activating Factor ◽

Test System ◽

Human Platelets ◽

Vacuum Filtration ◽

Reversible Aggregation ◽

The Individual ◽

20 Nm ◽

And Control

PAF is suggested to be of pathophysiological importance in a variety of diseases. Since platelets exhibit a reduced sensitivity to PAF after a contact with this agent, this behavior may be used as indicator of PAF released into the circulation. In extrinsic asthma, platelets show a diminished reaction to PAF after exposition of the patients to the antigen compared to their own platelets before exposition (Beer and von Felten, Adv. Inflamm. Res. 10:323,1986). We were therefore looking for a test system indicating directly whether platelets had been in contact with PAF.Preparation of PAF-desensitized platelets: Citrated PRP was placed in a cuvette of an aggregometer, and PAF was added in 10 portions at intervals of 10 sec (37oc, constant stirring) to a final concentration of 10 to 100 nM, depending on the individual sensitivity of each platelet preparation. Therby, only a minimal, completely reversible aggregation was registered without any release of serotonin (ST) or 3-thromboglobulin (BTG). Control platelets were pretreated with buffer instead of PAF. Both platelets preparations were kept at 37°C for 45 min. Whereas control platelets showed a secondary aggregation to PAF (5x conc. used for desensitization), PAF-pretreated piatelets were only reversibly aggregated.Sensitivity of PAF-desensitized and control platelets to other platelet agonists: No difference in aggregation, ST-or BTG-relea-se was observed after stimulation with several concentrations of ADP, collagen and arachidonate (p>0.05,n= 41).Binding of 3H-PAF to platelets: PAF-desensitized and control platelets were separated from plasma by filtration through sepharose CL-2B (Pharmacia) in hepes-buffered Tyrode’s solution. After incubation with 3H-PAF, platelets were washed on Whatman 934-AH filters (vacuum filtration). On desensitized and control platelets, we found 175±48 (mean±sd) and 231±70 3H-PAF molecules / platelet respectively after incubation with 5 nM ^h-PAF, 399±36 and 504±66 ^H-PAF molecules / platelet after incubation with 20 nM. In spite of a statistically significant reduction of PAF-binding after desensitization (p<0.01),the variability of PAF-binding between platelets of different individuals is too high to allow a discrimination of normal from PAF-desensitized platelets.

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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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Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽

10.1182/blood-2009-04-217240 ◽

2010 ◽

Vol 115 (1) ◽

pp. 97-106 ◽

Cited By ~ 45

Author(s):

Yacine Boulaftali ◽

Frédéric Adam ◽

Laurence Venisse ◽

Véronique Ollivier ◽

Benjamin Richard ◽

...

Keyword(s):

Ex Vivo ◽

Thrombus Formation ◽

Human Platelets ◽

Platelet Surface ◽

Type Plasminogen Activator ◽

Healthy Donors ◽

Protease Nexin ◽

Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

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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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