The Experts below are selected from a list of 684 Experts worldwide ranked by ideXlab platform
Chris Baylis - One of the best experts on this subject based on the ideXlab platform.
-
Resistance to renal damage by chronic nitric oxide synthase inhibition
2006Co-Authors: Aaron Erdely, Gary Freshour, Chris Baylis, C BaylisAbstract:Erdely, Aaron, Gary Freshour, and Chris Baylis. Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth Rat. Am J Physiol Regul Integr Comp Physiol 290: R66 -R72, 2006; doi:10.1152/ajpregu.00444.2005.-Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD Rats, whereas low-dose NOSI caused progression of CKD in WF Rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD Rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N G -nitro-L-arginine methyl ester (L-NAME) (150 and 300 mg/l for 6 -10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic L-NAME. SD given 150 mg/l L-NAME for 6 wk developed proteinuria and renal injury, whereas WF Rats receiving 150 mg/l L-NAME for 6 -10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD Rat. There was little impact on renal nitric oxide synthase expression with L-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF Rats, whereas WF Rats showed a much smaller increment in glomerular blood pressure compared with SD Rats. In conclusion, WF Rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD Rat. This protection may be associated with protection from glomerular hypertension. proteinuria; glomerular sclerosis; creatinine clearance; SpragueDawley THERE IS CLEAR EVIDENCE from animal studies that chronic nitric oxide synthase (NOS) inhibition (NOSI) causes dose-dependent hypertension and progressive kidney damage. Incomplete NOSI leads to modeRate systemic and glomerular hypertension, mild proteinuria, and modeRate focal and segmental glomerular sclerosis. Higher doses create an acceleRated model of hypertension and kidney damage, and the systemic and glomerular hypertension contribute to the increased severity of kidney damage (37). In addition, the potent vasoconstrictor effect of NO deficiency causes declines in renal function and the nonhemodynamic actions of NO deficiency include permissive mesangial growth and matrix overproduction, promoting glomerular sclerosis (21). These findings have clinical relevance, given the evidence that NO deficiency occurs in humans as a result of chronic kidney disease (CKD) (4, 6, 26 -28, 34). One site of systemic NO deficiency is undoubtedly associated with the widespread endothelial dysfunction seen in renal disease, at least in part due to elevations in the endogenous NOS inhibitor asymmetric dimethylarginine (7). In a variety of animal models of CKD, a decrease in total NO production has been reported as well as a reduced renal NOS abundance and activity The Wistar-Furth (WF) Rat is interesting in that it is not only resistant to renal mass reduction models of CKD (15, 16) but also to chronic puromycin-induced CKD (12), as well as mineralocorticoid-induced hypertension and renal injury METHODS For chronic NOSI studies. Studies were conducted on two strains of Rats, Sprague-Dawley (SD) and Wistar-Furth (WF) purchased from Harlan (Indianapolis, IN) and age-matched at 10 wk. Rats were divided into groups as shown in Metabolic cage collections of 24 h urines were made at baseline and at weekly intervals of chronic NOSI to measure urinary tota
-
resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar Furth Rat
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2006Co-Authors: Aaron Erdely, Gary Freshour, Chris BaylisAbstract:Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several mod...
-
protection against puromycin aminonucleoside induced chronic renal disease in the Wistar Furth Rat
American Journal of Physiology-renal Physiology, 2004Co-Authors: Aaron Erdely, Gary Freshour, Cheryl A Smith, Kevin Engels, Jean L Olson, Chris BaylisAbstract:The Wistar-Furth (WF) Rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) Rat, and protection was associated with preserved renal ni...
Aaron Erdely - One of the best experts on this subject based on the ideXlab platform.
-
Resistance to renal damage by chronic nitric oxide synthase inhibition
2006Co-Authors: Aaron Erdely, Gary Freshour, Chris Baylis, C BaylisAbstract:Erdely, Aaron, Gary Freshour, and Chris Baylis. Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth Rat. Am J Physiol Regul Integr Comp Physiol 290: R66 -R72, 2006; doi:10.1152/ajpregu.00444.2005.-Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD Rats, whereas low-dose NOSI caused progression of CKD in WF Rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD Rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N G -nitro-L-arginine methyl ester (L-NAME) (150 and 300 mg/l for 6 -10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic L-NAME. SD given 150 mg/l L-NAME for 6 wk developed proteinuria and renal injury, whereas WF Rats receiving 150 mg/l L-NAME for 6 -10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD Rat. There was little impact on renal nitric oxide synthase expression with L-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF Rats, whereas WF Rats showed a much smaller increment in glomerular blood pressure compared with SD Rats. In conclusion, WF Rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD Rat. This protection may be associated with protection from glomerular hypertension. proteinuria; glomerular sclerosis; creatinine clearance; SpragueDawley THERE IS CLEAR EVIDENCE from animal studies that chronic nitric oxide synthase (NOS) inhibition (NOSI) causes dose-dependent hypertension and progressive kidney damage. Incomplete NOSI leads to modeRate systemic and glomerular hypertension, mild proteinuria, and modeRate focal and segmental glomerular sclerosis. Higher doses create an acceleRated model of hypertension and kidney damage, and the systemic and glomerular hypertension contribute to the increased severity of kidney damage (37). In addition, the potent vasoconstrictor effect of NO deficiency causes declines in renal function and the nonhemodynamic actions of NO deficiency include permissive mesangial growth and matrix overproduction, promoting glomerular sclerosis (21). These findings have clinical relevance, given the evidence that NO deficiency occurs in humans as a result of chronic kidney disease (CKD) (4, 6, 26 -28, 34). One site of systemic NO deficiency is undoubtedly associated with the widespread endothelial dysfunction seen in renal disease, at least in part due to elevations in the endogenous NOS inhibitor asymmetric dimethylarginine (7). In a variety of animal models of CKD, a decrease in total NO production has been reported as well as a reduced renal NOS abundance and activity The Wistar-Furth (WF) Rat is interesting in that it is not only resistant to renal mass reduction models of CKD (15, 16) but also to chronic puromycin-induced CKD (12), as well as mineralocorticoid-induced hypertension and renal injury METHODS For chronic NOSI studies. Studies were conducted on two strains of Rats, Sprague-Dawley (SD) and Wistar-Furth (WF) purchased from Harlan (Indianapolis, IN) and age-matched at 10 wk. Rats were divided into groups as shown in Metabolic cage collections of 24 h urines were made at baseline and at weekly intervals of chronic NOSI to measure urinary tota
-
resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar Furth Rat
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2006Co-Authors: Aaron Erdely, Gary Freshour, Chris BaylisAbstract:Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several mod...
-
protection against puromycin aminonucleoside induced chronic renal disease in the Wistar Furth Rat
American Journal of Physiology-renal Physiology, 2004Co-Authors: Aaron Erdely, Gary Freshour, Cheryl A Smith, Kevin Engels, Jean L Olson, Chris BaylisAbstract:The Wistar-Furth (WF) Rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) Rat, and protection was associated with preserved renal ni...
Carl W. Jackson - One of the best experts on this subject based on the ideXlab platform.
-
Prolonged Bleeding Time With Defective Platelet Filopodia Formation in the Wistar Furth Rat
2016Co-Authors: E. Stenberg, Shirley A. Steward, Nancy K Hutson, Tamara I Pestina, Rosemary J. Barrie, Julie T. Arnold, Aparna K. Murti, Carl W. JacksonAbstract:Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) Rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskel-etal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (G30 minutes in 10 of 11 Rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggrega-tion, or the release reaction. Because aggregation to colla-gen and adenosine diphosphate were reported to be normal, we determined whether WF Rat platelets are defective i
-
Wistar Furth Rat megakaryocytes lack dense compartments and intercellular plaques membranous structures rich in cytoskeletal proteins
Cell Adhesion and Communication, 1998Co-Authors: Paula E Stenberg, Jay H Beckstead, Carl W. JacksonAbstract:Wistar Furth (WF) Rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF Rat megakaryocytes to those of Rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administRation, have revealed a previously unrecognized membrane structure in normal Rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar Rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF Rats. The sec...
-
prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth Rat
Blood, 1998Co-Authors: Paula E Stenberg, Shirley A. Steward, Nancy K Hutson, Tamara I Pestina, Rosemary J. Barrie, Julie T. Arnold, Aparna K. Murti, Carl W. JacksonAbstract:Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) Rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 Rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF Rat platelets are defective in their ability to adhere to substRates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal Rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF Rats.
-
abnormal subcellular distribution of myosin and talin in Wistar Furth Rat platelets
Blood, 1995Co-Authors: Tamara I Pestina, Carl W. Jackson, Paula E StenbergAbstract:The roles of most cytoskeletal proteins in platelet formation and function remain largely undefined. We earlier detected megakaryocyte membrane blebbing and a unique antigenic determinant associated with a missense mutation in the cytoskeletal protein, talin, in an animal model of hereditary macrothrombocytopenia, the Wistar Furth (WF) Rat, which led us to examine the distribution of talin and other cytoskeletal proteins in resting normal and WF Rat platelets. In contrast to the conclusions of an earlier ultrastructural analysis, our biochemical and ultrastructural immunogold studies indicate a significant membrane-association of talin in both resting normal and WF Rat platelets as found earlier for Rat megakaryocytes. Talin was associated with plasma membranes, membranes of the surface-connected canalicular system, and with alpha-granule membranes of both normal and WF Rat platelets, but as in WF megakaryocytes, talin was absent from the large membrane complexes of WF platelets. An even more striking difference was seen in the distribution of myosin in subcellular fractions of normal and WF Rat platelets sepaRated in density gradients, in which the proportion of myosin in the least dense WF Rat platelet membrane fraction was one half that in the same normal platelet fraction. This difference was balanced by a fourfold increase in myosin in the most dense WF Rat subcellular fraction, which is highly enriched for alpha-granules. These results support our hypothesis that the platelet abnormalities of the WF Rat are related to defects in the megakaryocyte-platelet cytoskeleton.
-
disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal Rats like those in human and Wistar Furth Rat hereditary macrothrombocytopenias
Journal of Cellular Physiology, 1995Co-Authors: Paula E Stenberg, T P Mcdonald, Carl W. JacksonAbstract:Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth Rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar Rat bone marrow was fixed and processed for transmission electron microscopy after VCR administRation alone, after 5-fluorouracil (5-FU) administRation alone, or after 5-FU followed by intravenous injection of 0.1-1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar Rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth Rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (approximately two-thirds of megakaryocytes) at VCR dosages of 0.75-1.0 mg/kg. The majority of megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted alpha-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstRate these abnormalities, with the exception of an increase in dense compartments. Platelets from Rats treated with VCR alone or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar Rats as well as untreated Wistar Furth Rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated Rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte alpha-granules.
Gary Freshour - One of the best experts on this subject based on the ideXlab platform.
-
Resistance to renal damage by chronic nitric oxide synthase inhibition
2006Co-Authors: Aaron Erdely, Gary Freshour, Chris Baylis, C BaylisAbstract:Erdely, Aaron, Gary Freshour, and Chris Baylis. Resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar-Furth Rat. Am J Physiol Regul Integr Comp Physiol 290: R66 -R72, 2006; doi:10.1152/ajpregu.00444.2005.-Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several models of CKD and maintain renal nitric oxide (NO) production compared with SD Rats, whereas low-dose NOSI caused progression of CKD in WF Rats. Here, we evaluate the impact of high-dose chronic NOSI in WF and SD Rats, as well as intrarenal responses to an acute pressor dose of NOSI in the normal WF. Rats were given N G -nitro-L-arginine methyl ester (L-NAME) (150 and 300 mg/l for 6 -10 wk) in the drinking water after an initial bolus tail vein injection. Both strains showed significant reductions in total NO production with chronic L-NAME. SD given 150 mg/l L-NAME for 6 wk developed proteinuria and renal injury, whereas WF Rats receiving 150 mg/l L-NAME for 6 -10 wk or 300 mg/l for 6 wk developed no proteinuria and minimal renal injury. Blood pressure was significantly elevated with chronic NOSI in both strains but was higher in the SD Rat. There was little impact on renal nitric oxide synthase expression with L-NAME, except that cortical endothelial nitric oxide synthase abundance increased in WF after 6 wk (150 mg/l). Micropuncture experiments with acute pressor NOSI resulted in similar increases in systemic blood pressure in SD and WF Rats, whereas WF Rats showed a much smaller increment in glomerular blood pressure compared with SD Rats. In conclusion, WF Rats do not develop renal injury after chronic NOSI at, or above, a dose that causes significant injury in the SD Rat. This protection may be associated with protection from glomerular hypertension. proteinuria; glomerular sclerosis; creatinine clearance; SpragueDawley THERE IS CLEAR EVIDENCE from animal studies that chronic nitric oxide synthase (NOS) inhibition (NOSI) causes dose-dependent hypertension and progressive kidney damage. Incomplete NOSI leads to modeRate systemic and glomerular hypertension, mild proteinuria, and modeRate focal and segmental glomerular sclerosis. Higher doses create an acceleRated model of hypertension and kidney damage, and the systemic and glomerular hypertension contribute to the increased severity of kidney damage (37). In addition, the potent vasoconstrictor effect of NO deficiency causes declines in renal function and the nonhemodynamic actions of NO deficiency include permissive mesangial growth and matrix overproduction, promoting glomerular sclerosis (21). These findings have clinical relevance, given the evidence that NO deficiency occurs in humans as a result of chronic kidney disease (CKD) (4, 6, 26 -28, 34). One site of systemic NO deficiency is undoubtedly associated with the widespread endothelial dysfunction seen in renal disease, at least in part due to elevations in the endogenous NOS inhibitor asymmetric dimethylarginine (7). In a variety of animal models of CKD, a decrease in total NO production has been reported as well as a reduced renal NOS abundance and activity The Wistar-Furth (WF) Rat is interesting in that it is not only resistant to renal mass reduction models of CKD (15, 16) but also to chronic puromycin-induced CKD (12), as well as mineralocorticoid-induced hypertension and renal injury METHODS For chronic NOSI studies. Studies were conducted on two strains of Rats, Sprague-Dawley (SD) and Wistar-Furth (WF) purchased from Harlan (Indianapolis, IN) and age-matched at 10 wk. Rats were divided into groups as shown in Metabolic cage collections of 24 h urines were made at baseline and at weekly intervals of chronic NOSI to measure urinary tota
-
resistance to renal damage by chronic nitric oxide synthase inhibition in the Wistar Furth Rat
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2006Co-Authors: Aaron Erdely, Gary Freshour, Chris BaylisAbstract:Chronic nitric oxide synthase inhibition (NOSI) causes chronic kidney disease (CKD) in the Sprague Dawley (SD) Rat. We previously showed that the Wistar-Furth (WF) Rats are resistant to several mod...
-
protection against puromycin aminonucleoside induced chronic renal disease in the Wistar Furth Rat
American Journal of Physiology-renal Physiology, 2004Co-Authors: Aaron Erdely, Gary Freshour, Cheryl A Smith, Kevin Engels, Jean L Olson, Chris BaylisAbstract:The Wistar-Furth (WF) Rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) Rat, and protection was associated with preserved renal ni...
Paula E Stenberg - One of the best experts on this subject based on the ideXlab platform.
-
Wistar Furth Rat megakaryocytes lack dense compartments and intercellular plaques membranous structures rich in cytoskeletal proteins
Cell Adhesion and Communication, 1998Co-Authors: Paula E Stenberg, Jay H Beckstead, Carl W. JacksonAbstract:Wistar Furth (WF) Rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF Rat megakaryocytes to those of Rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administRation, have revealed a previously unrecognized membrane structure in normal Rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar Rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF Rats. The sec...
-
prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth Rat
Blood, 1998Co-Authors: Paula E Stenberg, Shirley A. Steward, Nancy K Hutson, Tamara I Pestina, Rosemary J. Barrie, Julie T. Arnold, Aparna K. Murti, Carl W. JacksonAbstract:Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) Rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 Rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF Rat platelets are defective in their ability to adhere to substRates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal Rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF Rats.
-
abnormal subcellular distribution of myosin and talin in Wistar Furth Rat platelets
Blood, 1995Co-Authors: Tamara I Pestina, Carl W. Jackson, Paula E StenbergAbstract:The roles of most cytoskeletal proteins in platelet formation and function remain largely undefined. We earlier detected megakaryocyte membrane blebbing and a unique antigenic determinant associated with a missense mutation in the cytoskeletal protein, talin, in an animal model of hereditary macrothrombocytopenia, the Wistar Furth (WF) Rat, which led us to examine the distribution of talin and other cytoskeletal proteins in resting normal and WF Rat platelets. In contrast to the conclusions of an earlier ultrastructural analysis, our biochemical and ultrastructural immunogold studies indicate a significant membrane-association of talin in both resting normal and WF Rat platelets as found earlier for Rat megakaryocytes. Talin was associated with plasma membranes, membranes of the surface-connected canalicular system, and with alpha-granule membranes of both normal and WF Rat platelets, but as in WF megakaryocytes, talin was absent from the large membrane complexes of WF platelets. An even more striking difference was seen in the distribution of myosin in subcellular fractions of normal and WF Rat platelets sepaRated in density gradients, in which the proportion of myosin in the least dense WF Rat platelet membrane fraction was one half that in the same normal platelet fraction. This difference was balanced by a fourfold increase in myosin in the most dense WF Rat subcellular fraction, which is highly enriched for alpha-granules. These results support our hypothesis that the platelet abnormalities of the WF Rat are related to defects in the megakaryocyte-platelet cytoskeleton.
-
disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal Rats like those in human and Wistar Furth Rat hereditary macrothrombocytopenias
Journal of Cellular Physiology, 1995Co-Authors: Paula E Stenberg, T P Mcdonald, Carl W. JacksonAbstract:Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth Rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar Rat bone marrow was fixed and processed for transmission electron microscopy after VCR administRation alone, after 5-fluorouracil (5-FU) administRation alone, or after 5-FU followed by intravenous injection of 0.1-1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar Rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth Rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (approximately two-thirds of megakaryocytes) at VCR dosages of 0.75-1.0 mg/kg. The majority of megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted alpha-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstRate these abnormalities, with the exception of an increase in dense compartments. Platelets from Rats treated with VCR alone or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar Rats as well as untreated Wistar Furth Rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated Rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte alpha-granules.
-
A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth Rat.
Blood, 1992Co-Authors: Carl W. Jackson, Shirley A. Steward, Nancy K Hutson, Paula E StenbergAbstract:Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF Rat platelets and absorbed with Wistar Rat platelets recognized a major 235-Kd band, and minor bands of WF Rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans Rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as Rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of Rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF Rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar Rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF Rats suggests that talin is abnormal in this Rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.
