- Email: [email protected]
Apoptotic vascular endothelial cells become procoagulant
314
Daily Measurements of Blood CD34+ Cells After Stem Cell Mobilization Predict Stem Cell Yield and Posttransplant Hematopoietic Recovery. Remes K, Matinlauri I, Grenman S, etaL J Hematotherapy 6:13-19, 1997. This single institution study provides significant additional data to support the use of CD34 cell counting in the management of an autologous stem cell transplant program. The authors describe their experience with 60 patients undergoing autologous transplant for a variety of conditions including myeloma, non-Hodgkin's lymphoma, breast cancer, and ovarian cancer. Patients underwent a fairly typical mobilization regimen of cyclophosphamide plus G-CSF/GM-CSF at 5 pg/kg for at least 4 days followed by a 10 liter apheresis harvest. Daily CD34 cell measurements were obtained and the results correlated with clinical outcomes. CD34 measurements on peripheral blood were done by flow cytometry using an "in-house" assay that used phycoerythrin-conjugated HPCA-2 clone to stain mononuclear cells that had been isolated using a commercial preparation (Leukoprep). Absolute counts were obtained by multiplying the proportion of CD34+ mononuclears by the mononuclear count obtained with a Technicon H2 analyzer. The results showed quite a good correlation (r = 0.9) in the linear relationship between the pre-apheresis blood CD34 absolute count and the apheresis CD34 content expressed as 106 cells/kg recipient. There were also good hyperbolic relationships between the number of infused CD34 cells (expressed as 106/kg recipient) and the days to platelet recovery or the days to hemoglobin recovery. This latter data suggested that the threshold value for good recovery was 4 • 106 CD34+/kg recipient, which is in close agreement with other studies. They showed that patients receiving this dose of cells (or higher) had a slightly shorter length of stay and used less blood products than patients receiving fewer cells. Of real practical value, the authors then exmnined measurements of CD34 cell concentration in the patient before stem cell harvest as a predictor of collection of a threshold quantity of stem cells. They found that patients who present to the apheresis service with less than 20 CD34/gL never achieved an adequate cell collection even with processing up to 50 L of blood. Patients who mobilized to a level of 20 to 50 CD34/pL required a median of 3 procedures (range 2-5) to achieve an adequate stem cell dose and only 1 of 15 procedures yielded fewer than 1.5 • 106 CD34/kg in the harvest. For patients who present with more than 50 CD34/gL, none of 35 apheresis harvest yielded fewer than 1.5 • 106 CD34/kg and patients required a median of 1 procedure to achieve a therapeutic dose of cells. As standardization of CD34 counting continues to improve, CD34 measurements will be increasingly reliable. This article gives insight into how these measurements can be used to advantage ~n the care of patients with cancer.
Deficient Activity of von Willebrand Factor-Cleaving Protease in Chronic Relapsing Thrombotic Thrombocytopenic Purpura. Furlan M, Robles R, Solenthaler M, et aL Blood 89:3097-3103, 1997. Patients with chronic relapsing TTP are being increasingly identified and represent a difficult and challenging treatment problem for transfusion medicine. Although the interplay of inheritance and environment have long been considered in discussions of TTP, this report lends more experimental evidence to a genetic predisposition to TTP. The release of
CURRENT ABSTRACTS ultralarge multimers of vWF has been previously shown in patients with TTP and has been said to promote intravascular platelet thrombi. In this article, the authors examined the plasma of four individuals with relapsing TTP (including two brothers) for evidence of a deficiency of a protease capable of cleaving high molecular weight forms of vonWillebrand's mulfimers. Von Willebrand factor multimer analysis was performed using standard electrophoresis and a rabbit antiserum against human vWR The activity of the vWF-cleaving protease was assayed at low ionic strength in the presence of urea. Care was taken to avoid contaminating the assay with external proteases and a serine protease inhibitor was added to block the natural serine protease activity of plasma. Pooled normal human plasma samples and samples from the four subjects were diluted and tested for their ability to cleave a measured amount of purified vWF added to the assay. Because decreased ability to cleave vWF could be caused by an inhibitor in the patients' plasma, the presence of inhibitors was investigated by mixing patient samples with normal plasma samples and repeating the assay for vWF cleavage. Each of the patient samples showed absent vWF-cleaving protease activity. There was no evidence for an inhibitor to account for the deficient protease activity. Deficiency of an enzyme required to cleave vWF has been previously proposed as a potential mechanism for TTP. This article supports this concept and provides a rational for the use of replacement therapy (infusion of plasma) in the treatment of TTP. Of particular interest, it also suggests that individuals may have a genetic predisposition to TTP (and perhaps other disorders mediated by high molecular forms of vWF) as a result of the absence or deficiency of the cleaving protease. The combination of such a genetic predisposition and an environment insult that initiates endothelial damage may represent the "one-two punch" needed to initiate this serious thrombotic syndrome. Research into the identification of the protease may one day lead to improved diagnosis and a replacement therapy which is more specific than that of whole plasma.
Apoptotic Vascular Endothelial Cells Become Procoagulant. Bombeli T, Karsan A, Tait JF, Harlan JM. Blood 89:2429-2442, 1997. Programmed cell death is increasingly recognized as a widely used cellular response to injury. This study comes from a leading research laboratory in the field of vascular biology and shows that apoptosis of vascular endothelial cells provides a procoagulant signal. Because of the recent finding that endothelial cell apoptosis occurs in thrombotic thrombocytopenic purpura (TIP), the results of this study may help explain the thrombotic nature of TTP. The investigators studied human umbilical vein endothelial cells and induced apoptosis by treatment with staurosporine--a protein kinase inhibitor. Procoagulant signals were assessed in several ways including flow cytometric determination of binding of annexin V to endothelial cells. Annexin V is a marker for the phospholipid inversions and exposure of phosphotidylserine which occurs in cell membranes that are activated to procoagulant signals. In addition, the endothelial cells displayed more intrinsic ability to activate coagulation factor ten and increased the production of thrombin in citrated plasma. Importantly (and not unexpectedly) apoptotic endothelial cells lost significant expression of antithrombotic cell surface structures including
CURRENT ABSTRACTS
thrombomodulin, heparan sulfates, and tissue-factor pathway inhibitor. Widespread endothelial injury may accompany a large number of conditions recognized as hypercoagulation states including heparin-associated thrombocytopenia, disseminated intravascular coagulation, idiopathic TTP, drug-induced TTP, systemic
315
lupus, adult respiratory distress syndrome, and shock/sepsis. In addition, local endothelial damage is likely to accompany a wide variety of disorders that produce focal tissue inflammation. This study suggests that endothelial apoptosis may represent an important possible underlying theme connecting inflammation and coagulation.
Daily Measurements of Blood CD34+ Cells After Stem Cell Mobilization Predict Stem Cell Yield and Posttransplant Hematopoietic Recovery. Remes K, Matinlauri I, Grenman S, etaL J Hematotherapy 6:13-19, 1997. This single institution study provides significant additional data to support the use of CD34 cell counting in the management of an autologous stem cell transplant program. The authors describe their experience with 60 patients undergoing autologous transplant for a variety of conditions including myeloma, non-Hodgkin's lymphoma, breast cancer, and ovarian cancer. Patients underwent a fairly typical mobilization regimen of cyclophosphamide plus G-CSF/GM-CSF at 5 pg/kg for at least 4 days followed by a 10 liter apheresis harvest. Daily CD34 cell measurements were obtained and the results correlated with clinical outcomes. CD34 measurements on peripheral blood were done by flow cytometry using an "in-house" assay that used phycoerythrin-conjugated HPCA-2 clone to stain mononuclear cells that had been isolated using a commercial preparation (Leukoprep). Absolute counts were obtained by multiplying the proportion of CD34+ mononuclears by the mononuclear count obtained with a Technicon H2 analyzer. The results showed quite a good correlation (r = 0.9) in the linear relationship between the pre-apheresis blood CD34 absolute count and the apheresis CD34 content expressed as 106 cells/kg recipient. There were also good hyperbolic relationships between the number of infused CD34 cells (expressed as 106/kg recipient) and the days to platelet recovery or the days to hemoglobin recovery. This latter data suggested that the threshold value for good recovery was 4 • 106 CD34+/kg recipient, which is in close agreement with other studies. They showed that patients receiving this dose of cells (or higher) had a slightly shorter length of stay and used less blood products than patients receiving fewer cells. Of real practical value, the authors then exmnined measurements of CD34 cell concentration in the patient before stem cell harvest as a predictor of collection of a threshold quantity of stem cells. They found that patients who present to the apheresis service with less than 20 CD34/gL never achieved an adequate cell collection even with processing up to 50 L of blood. Patients who mobilized to a level of 20 to 50 CD34/pL required a median of 3 procedures (range 2-5) to achieve an adequate stem cell dose and only 1 of 15 procedures yielded fewer than 1.5 • 106 CD34/kg in the harvest. For patients who present with more than 50 CD34/gL, none of 35 apheresis harvest yielded fewer than 1.5 • 106 CD34/kg and patients required a median of 1 procedure to achieve a therapeutic dose of cells. As standardization of CD34 counting continues to improve, CD34 measurements will be increasingly reliable. This article gives insight into how these measurements can be used to advantage ~n the care of patients with cancer.
Deficient Activity of von Willebrand Factor-Cleaving Protease in Chronic Relapsing Thrombotic Thrombocytopenic Purpura. Furlan M, Robles R, Solenthaler M, et aL Blood 89:3097-3103, 1997. Patients with chronic relapsing TTP are being increasingly identified and represent a difficult and challenging treatment problem for transfusion medicine. Although the interplay of inheritance and environment have long been considered in discussions of TTP, this report lends more experimental evidence to a genetic predisposition to TTP. The release of
CURRENT ABSTRACTS ultralarge multimers of vWF has been previously shown in patients with TTP and has been said to promote intravascular platelet thrombi. In this article, the authors examined the plasma of four individuals with relapsing TTP (including two brothers) for evidence of a deficiency of a protease capable of cleaving high molecular weight forms of vonWillebrand's mulfimers. Von Willebrand factor multimer analysis was performed using standard electrophoresis and a rabbit antiserum against human vWR The activity of the vWF-cleaving protease was assayed at low ionic strength in the presence of urea. Care was taken to avoid contaminating the assay with external proteases and a serine protease inhibitor was added to block the natural serine protease activity of plasma. Pooled normal human plasma samples and samples from the four subjects were diluted and tested for their ability to cleave a measured amount of purified vWF added to the assay. Because decreased ability to cleave vWF could be caused by an inhibitor in the patients' plasma, the presence of inhibitors was investigated by mixing patient samples with normal plasma samples and repeating the assay for vWF cleavage. Each of the patient samples showed absent vWF-cleaving protease activity. There was no evidence for an inhibitor to account for the deficient protease activity. Deficiency of an enzyme required to cleave vWF has been previously proposed as a potential mechanism for TTP. This article supports this concept and provides a rational for the use of replacement therapy (infusion of plasma) in the treatment of TTP. Of particular interest, it also suggests that individuals may have a genetic predisposition to TTP (and perhaps other disorders mediated by high molecular forms of vWF) as a result of the absence or deficiency of the cleaving protease. The combination of such a genetic predisposition and an environment insult that initiates endothelial damage may represent the "one-two punch" needed to initiate this serious thrombotic syndrome. Research into the identification of the protease may one day lead to improved diagnosis and a replacement therapy which is more specific than that of whole plasma.
Apoptotic Vascular Endothelial Cells Become Procoagulant. Bombeli T, Karsan A, Tait JF, Harlan JM. Blood 89:2429-2442, 1997. Programmed cell death is increasingly recognized as a widely used cellular response to injury. This study comes from a leading research laboratory in the field of vascular biology and shows that apoptosis of vascular endothelial cells provides a procoagulant signal. Because of the recent finding that endothelial cell apoptosis occurs in thrombotic thrombocytopenic purpura (TIP), the results of this study may help explain the thrombotic nature of TTP. The investigators studied human umbilical vein endothelial cells and induced apoptosis by treatment with staurosporine--a protein kinase inhibitor. Procoagulant signals were assessed in several ways including flow cytometric determination of binding of annexin V to endothelial cells. Annexin V is a marker for the phospholipid inversions and exposure of phosphotidylserine which occurs in cell membranes that are activated to procoagulant signals. In addition, the endothelial cells displayed more intrinsic ability to activate coagulation factor ten and increased the production of thrombin in citrated plasma. Importantly (and not unexpectedly) apoptotic endothelial cells lost significant expression of antithrombotic cell surface structures including
CURRENT ABSTRACTS
thrombomodulin, heparan sulfates, and tissue-factor pathway inhibitor. Widespread endothelial injury may accompany a large number of conditions recognized as hypercoagulation states including heparin-associated thrombocytopenia, disseminated intravascular coagulation, idiopathic TTP, drug-induced TTP, systemic
315
lupus, adult respiratory distress syndrome, and shock/sepsis. In addition, local endothelial damage is likely to accompany a wide variety of disorders that produce focal tissue inflammation. This study suggests that endothelial apoptosis may represent an important possible underlying theme connecting inflammation and coagulation.