calpain and Purpura--Thrombotic-Thrombocytopenic

Thrombotic thrombocytopenic purpura is an uncommon disorder, but it continues to be of considerable interest. The disease mechanisms are unclear and the aetiology is unknown. Perhaps most enigmatic of all, the mode of action of plasma therapy, which successfully induces remission in about two-thirds of cases, is wholly inexplicable. There are currently several areas of debate on the subject of thrombotic thrombocytopenic purpura. This paper addresses these points of contention: the definition of the disease, its distinction from haemolytic uraemic syndrome, the nature of the platelet aggregating factors in the plasma of patients with acute disease, the importance of the abnormalities of von Willebrand's factor observed in the acute and quiescent phases of the disease, the nature of the factor in normal plasma that induces remission, and the possible causes of the observed superiority of plasma exchange combined with plasma infusion, over plasma infusion alone.

Topics: Calpain; Hemolytic-Uremic Syndrome; Humans; Purpura, Thrombotic Thrombocytopenic; von Willebrand Factor

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) is not well understood. Recent studies have described a platelet aggregating factor which has been characterized as a calcium-dependent cysteine protease (calpain) in patients with TTP. A type of TTP, sometimes called secondary TTP, has been associated with bone marrow transplantation (BMT). However, unlike primary adult TTP, BMT-TTP has important differences and often does not respond well to plasma exchange. We describe the measurement of calpain activity in a group of BMT patients (with and without the clinical syndrome of transplant-associated TTP). Calpain was measured using a functional assay (14C-serotonin platelet release with inhibition by the cysteine protease inhibitor, leupeptin) in the sera of patients following autologous (auto) or allogeneic (allo) BMT. We also independently diagnosed and graded the BMT-TTP on the day of blood sampling using a scale that related to the percentage schistocytes and lactic dehydrogenase level. Calpain activity was detected in 1/8 (13%) grade 0-1 (6 auto, 2 allo); 6/16 (38%) grade 2 (3 auto, 13 allo) 9/16 (56%) grade 3 (2 auto, 14 allo) and 8/8 (100%) grade 4 BMT-TTP. Pre-BMT samples were tested in 10 allo-BMT patients who had positive calpain results post-BMT. One patient gave positive results before the transplant. This patient developed grade 4 BMT-TTP (day 24 post-BMT) and died despite apheresis. Positive calpain results were highly associated with neurologic symptoms, P < 0.001. Nineteen of 24 (79%) patients with positive results had neurologic symptoms compared to three of 21 (14%) patients with negative results. In conclusion, calpain was detected in half of the BMT patients with mild to moderate BMT-TTP (grades 2-3) and was uniformly found in those with severe (grade 4) BMT-TTP. Typically the calpain activity develops as TTP complicates the transplant process. It is unknown whether calpain contributes to the pathogenesis of this disorder, or is a secondary event.

Topics: Adult; Bone Marrow Transplantation; Calpain; Graft vs Host Disease; Humans; Purpura, Thrombotic Thrombocytopenic

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombocytopenia and schistocytic haemolytic anaemia. The majority of patients have normal coagulation parameters including the activated partial thromboplastin time (APTT). An intracellular cysteine protease, calpain, has been found in the plasma of many patients with acute TTP, and we hypothesize that it participates in the pathogenesis of the disorder. However, certain aspects of the disorder remain unresolved. For example, high molecular weight kininogen (HK) is one of the primary plasma inhibitors of calpain, and it also can act as a substrate for calpain. Consequently, one might anticipate that the HK could be defective or altered in TTP. In this report we describe the analysis of HK in plasma from live patients with acute TTP and following recovery. The HK was studied immunogenically and functionally. We observed that the HK in plasma samples from patients with acute TTP was proteolysed. This degradation was not observed in remission samples from the same patients. However, both acute and remission TTP samples had normal HK coagulant activity (acute samples, x = 0.84 +/- 0.26 U/ml; remission samples, x = 0.89 +/- 0.21 U/ml; control samples, x = 0.87 +/- 0.05 U/ml). Although the TTP plasmas were able to inhibit calpain activity, less inhibition activity was found in the acute samples (acute: mean inhibition 71 +/- 2.4%; remission: mean 92 +/- 2.1%; control samples: mean 93 +/- 5.4%; P < 0.001). Normal HK treated with calpain also had reduced calpain inhibition capacity (mean 58%). This study suggests that although HK is proteolysed during acute TTP, the proteolysis occurs without a major loss in the coagulation function or depletion of the protease inhibitory activity of HK.

Topics: Acute Disease; Blood Coagulation; Calpain; Humans; Immunoblotting; Kininogens; Purpura, Thrombotic Thrombocytopenic

Thrombotic thrombocytopenic purpura (TTP) is an uncommon disorder characterized by thrombocytopenia, schistocytic haemolytic anaemia, fever, neurologic lesions, and renal failure. A platelet aggregating factor has been postulated to be responsible for this disorder, but its precise identity remains debated. Two different groups of investigators have provided evidence that the platelet aggregating factor is a cysteine proteinase. We have suggested that it was calpain, whereas others have suggested that it was calpain, whereas others have suggested that it was cathepsin L. To help resolve this issue, we have studied the platelet activating activity found in the acute serum samples from 10 different TTP patients as well as purified calpain and cathepsin L. The TTP activity was measured functionally (platelet serotonin release assay and casein lysis assay) and antigenically (immunodepletion using anti-calpain and anti-cathepsin L antibodies and antigenic analysis using SDS PAGE). The TTP serum paralleled the activity of the purified calpain and had optimal pH activity of 7.5. The purified cathepsin L activity had optimal activity at low pH (5.5) and activity was no longer measurable at pH 7.5. Similarly, a specific cathepsin L inhibitor (Z-Phe-Phe-CHN2) had no effect on the activity of the TTP samples nor on purified calpain, but it did abolish the activity activity of purified cathepsin L. The platelet activating of the TTP samples was detectable in the microparticle pellet following ultracentrifugation of TTP serum, and could be immunodepleted using antibodies to calpain but not to cathepsin L. These studies indicate that the microparticle-associated platelet activating factor in TTP corresponds to calpain.

Topics: Blood Platelets; Calpain; Cathepsins; Humans; Purpura, Thrombotic Thrombocytopenic; Radioimmunoprecipitation Assay; Serotonin; Ultracentrifugation

Circulating platelet-aggregating activities have been described by several authors. In a previous study we have confirmed the presence in serum and plasma from patients with thrombotic thrombocytopenic purpura (TTP) of a platelet aggregating cysteine proteinase (PACP). This activity differed in that, among the class of thiol enzymes, it showed characteristics of a lysosomal cathepsin rather than of a cytoplasmic calpain. To further investigate the enzymatic properties of this PACP we have designed a study to evaluate the dependence of the activity on Ca++. Calcium-dependence is a property of calpains but not of cathepsins. A proteolytic assay was set up and conducted with and without Ca++. The release of acid-soluble peptide from denatured human globin promoted by TTP samples and standards was fluorimetrically measured. The results show that TTP samples were equally active with and without Ca++ similar to standard papain (the cathepsin B-like proteinase), whereas standard calpain only acted with Ca++. An inhibition study performed by the proteolytic assay confirmed the same pattern of sensitivity of PACP to a series of specific cystein proteinase inhibitors previously shown by the proaggregating assay. The enzymatic behaviour of PACP of TTP resembled a lysosomal (cathepsins) rather than a cytoplasmic (calpains) cysteine proteinase.

Topics: Calcium; Calpain; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Papain; Platelet Aggregation; Purpura, Thrombotic Thrombocytopenic

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.

Topics: Blood Platelets; Calpain; Humans; Immunoblotting; Kinetics; Molecular Weight; Purpura, Thrombotic Thrombocytopenic; Serotonin; Subcellular Fractions; Ultrafiltration

We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.

Topics: Binding, Competitive; Blood Platelets; Calpain; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Humans; Platelet Aggregation; Platelet Membrane Glycoproteins; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Factor

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.

Topics: Blood Platelets; Calpain; Glycoproteins; Humans; Kinetics; Platelet Aggregation; Protease Inhibitors; Purpura, Thrombotic Thrombocytopenic; Serotonin