Role of natural killer cells in tumor growth and metastasis: C57BL/6 normal and beige mice.

The role of natural killer (NK) cells in tumor growth and metastasis was studied in syngeneic normal and beige inbred C57BL/6 mice. Mice with the beige point mutation have been shown to be deficient in nonstimulated NK activity. Tumor-passaged B16 malignant melanoma cells were refractory to NK activity as determined by in vitro assay, but after in vitro culture they became sensitive to NK activity. The NK-insensitive B16 tumor grew and metastasized similarly in normal and beige mice. However, the NK-sensitive B16 tumors grew more slowly and produced fewer metastases in normal mice than in NK-deficient beige mice. Activation of NK cells by lymphocytic choriomeningitis virus infection decreased the rate of growth and number of metastases of both NK-sensitive and NK-insensitive tumors in both normal and beige mice. These results suggest the importance of NK cells as a determinant of tumor growth and metastasis.

Studies of platelet-bound and serum platelet-bindable immunoglobulins in dogs with idiopathic thrombocytopenic purpura.

Canine idiopathic thrombocytopenic purpura (ITP) is clinically analogous to chronic ITP in human beings. The objective of this study was to investigate the pathogenesis of canine ITP by determining whether immunoglobulins bound to the surface of platelets from dogs with ITP (platelet-bound immunoglobulins) were directed against host platelet antigen and whether platelet glycoproteins (GP) IIb and IIIa were target antigens in dogs with ITP. Thirty-two dogs with ITP were studied. Increased platelet-bound immunoglobulin concentrations were detected in 30 cases (94%), and increased concentrations of serum platelet-bindable immunoglobulins were detected in 11 cases (34%). Immunoglobulins eluted from the surface of platelets from dogs with ITP bound to homologous normal canine platelets in 11 of 19 cases (58%). Immunoglobulins against platelet membrane GP IIb and/or IIIa were detected in serum from four of 17 affected dogs. This study provides evidence that immunoglobulins bound to surface of platelets from some dogs with ITP are directed against host platelet antigens and that platelet target antigens are, in some cases, GP IIb and IIIa. This supports the hypothesis that canine ITP is an autoimmune disease, similar to the pathogenesis of chronic ITP in human beings.

Brain serotonin concentration and crude synaptosomal uptake in mice with the Chediak-Higashi syndrome.

The Chediak-Higashi syndrome is characterized by a serotonin platelet defect and neuronal dysfunction. Whole blood serotonin concentration, serotonin brain concentration, and synaptosomal uptake of serotonin were determined in mice with the syndrome. While brain serotonin uptake in the affected mice was not significantly different from that in nonaffected mice, whole blood serotonin concentration was markedly reduced. These data suggest that in human neuropathies with platelet serotonin defect, a parallel neuronal serotonin disorder may not be assumed.

[3H]-imipramine binding sites in fawn-hooded rats.

The existence of high-affinity [3H]-imipramine recognition sites was demonstrated in membranes prepared from the cerebral cortex, hypothalamus and platelets obtained from fawn-hooded rats. The Bmax and Kd values for [3H]-imipramine binding to cerebral cortical membranes were virtually identical to those obtained with cortical membrane preparations of Sprague-Dawley rats. An NBR strain of rats, genetically related to fawn-hooded rats, was found to have significantly higher levels of [3H]-imipramine binding sites in cerebral cortical membranes when compared to fawn-hooded and Sprague-Dawley rats. All four strains of rats examined possessed extremely high densities of [3H]-imipramine binding sites in a purified platelet membrane fraction. These results do not support the finding of others that the cerebral cortex and platelets of fawn-hooded rats are virtually devoid of [3H]-imipramine binding sites.

Evaluation of the buccal bleeding time and platelet glass bead retention as assays of hemostasis in the dog: the effects of acetylsalicylic acid, warfarin and von Willebrand factor deficiency.

The study evaluated two hemostatic assays in the dog, a modified version of the buccal mucosal bleeding time (BMBT) and the platelet glass bead retention (PR), to describe the aspects of hemostasis measured by these assays. Von Willebrand factor (vWf)-deficient Doberman pinscher dogs were used in evaluating the effects of altered platelet adhesion. Normal dogs were treated with either acetylsalicylic acid (ASA) or warfarin to evaluate the effects of altered platelet aggregation and coagulation. There was significant prolongation of the BMBT and reduction of the PR in vWf-deficient dogs as compared to normal dogs. In ASA treated dogs the BMBT was slightly prolonged; the PR was significantly reduced. The change in ASA-induced BMBT did not correlate with the sensitivity of the dog platelets to arachidonic acid. In warfarin treated dogs there was no change in the BMBT; however, the PR was significantly reduced. The BMBT is a test of hemostasis that is sensitive to platelet adhesion and aggregation deficits. The PR is useful in detecting general abnormalities in hemostasis including platelet adhesion defects due to reduced vWf.

Primary and secondary lysosomes in megakaryocytes and platelets from cattle with the Chediak-Higashi syndrome.

The ultrastructure of lysosomes from megakaryocytes (MK) and platelets of cattle with the Chediak-Higashi syndrome (CHS) was characterized using acid phosphatase histochemistry with beta-glycerophosphate as substrate and cerium as a capturing agent. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules in MK at all stages of maturation. Morphometric analysis of the diameter of each lysosome was performed on MK from CHS cattle and compared to MK from normal cattle. Lysosomes in CHS MK were neither enlarged nor different with respect to classification as secondary lysosomes, which composed 35% of the lysosomes in CHS MK. Lysosomes were demonstrated in 22% of the CHS platelet sections and appeared similar to those from normal cattle, 56% of them being classified as secondary lysosomes. Why lysosomes are not enlarged in bovine CHS MK and platelets, whereas they are enlarged in most other cell types, remains unknown.

Demonstration of secondary lysosomes in bovine megakaryocytes and platelets using acid phosphatase cytochemistry with cerium as a trapping agent.

The ultrastructure of lysosomes from bovine megakaryocytes (MK) and platelets was characterized using acid phosphatase cytochemistry with beta-glycerophosphate as substrate and cerium as a trapping agent. The technique was easily reproducible; cerium-phosphate precipitates were uniform, readily visualized, and there was a virtual absence of nonspecific reaction product. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules of less than 50 nm to 650 nm diameters in MK at all stages of maturation. Forty percent of the MK lysosomes contained inclusions of variable shapes, sizes and electron-density and were classified as secondary lysosomes. Twenty-four percent of the platelet sections contained acid phosphatase-positive granules. Fifty-four percent of these were secondary lysosomes. This is the initial report demonstrating secondary lysosomes in either resting MK or platelets using acid phosphatase cytochemistry. These findings suggest that MK and platelet lysosomes have an intracellular function in resting MK and platelets.

Platelet function, size and yield in whole blood and in platelet-rich plasma prepared using differing centrifugation force and time in domestic and food-producing animals.

The effects of centrifugation force and time upon platelets function, mean platelet volume and platelet yield were compared with whole blood platelet counts and size in citrated blood samples from the bovine, canine, caprine, equine, feline, ovine and porcine species. The results were similar, for a given species, irregardless of sample volume. Bovine, caprine, feline and ovine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine, equine and porcine platelet yields and mean platelet volumes were maximal when platelet-rich plasma was prepared using shorter centrifugation times and higher gravitational forces. Platelet aggregation to adenosine diphosphate or arachidonic acid was not effected by the method of platelet-rich plasma preparation in bovine, caprine, feline, ovine or porcine platelets. Equine platelet aggregation was maximal when platelet-rich plasma was prepared using longer centrifugation times and lower gravitational forces. Canine platelet aggregation, particularly arachidonic acid-induced aggregation, was maximal when platelet-rich plasma was prepared using short centrifugation times and higher gravitational forces. It appeared that the effects of centrifugation parameters upon platelet yield depended upon the relative difference between platelet and red blood cell volumes.

Prolonged bleeding time of Chediak-Higashi cats corrected by platelet transfusion.

Cats with the Chediak-Higashi syndrome (CHS) have a platelet storage pool deficiency (SPD). Ten CHS cats were transfused with a concentrate of 51Cr-labeled platelets prepared from normal donor cats. One hour after transfusion, the donor platelet count in CHS recipient cats was 40,000-60,000/microliters. Bleeding time before transfusion was 9.1 +/- 3.0 min. When donor platelet count in CHS cats was 50,000/microliters, bleeding time was 1.7 +/- 0.2 min. Bleeding time of normal cats was 1.4 +/- 0.3 min. Bleeding time increased to 3.3 +/- 0.2 min and to 5.3 +/- 0.2 min when the platelet count was 30,000/microliters, and 15,000/microliters, respectively. The close inverse relationship between bleeding time and number of donor platelets in CHS cats (r = -0.92), suggests that prolonged bleeding time is due to a platelet abnormality, that platelet transfusion can effectively correct prolonged bleeding time in an animal model of platelet SPD and that CHS cats may be an appropriate animal model to evaluate hemostatic capabilities of transfused platelets.

Rat platelet aggregation: strain and stock variations.

Platelet aggregation induced by ADP, arachidonic acid, and collagen was monitored in rats from two stocks, WSU-SD and CD, and from three strains, Lewis, Holtzman, and NBR. ADP-induced aggregation did not vary between the WSU-SD, CD, Lewis, Holtzman, and NBR rats. In contrast, the response to AA and collagen depended upon the stock or strain of rat. Platelets from the Holtzman and especially the NBR were much more sensitive to AA than were those from the other strains. At 0.25 mM AA, 7 of 8 NBR rats and 5 of 8 Holtzman rats aggregated irreversibly, while only 1 in 8 WSU-SD, CD, and Lewis rats aggregated irreversibly at that concentration. Collagen-induced aggregation reflected that to AA. The possible relationship between the variation in platelet aggregation and sympathoadrenal activity is discussed.

Anesthetics and anticoagulants used in the preparation of rat platelet-rich-plasma alter rat platelet aggregation.

Aggregation of platelets in heparin- and citrate-anticoagulated platelet-rich-plasma (PRP) from rats anesthetized with methoxyflurane (M), diethyl ether (E), acepromazine/ketamine (A/K), or sodium pentobarbital (P) is described, as are platelet counts. Platelet counts were highest in heparin- or citrate-PRP from E and A/K anesthetized rats. Collagen and arachidonic acid (AA) induced aggregation in heparin-PRP only, and ADP induced greater aggregation in heparin-PRP than in citrate-PRP. Differences between citrate-PRP and heparin-PRP are probably due to citrate inhibition of platelet aggregation, since addition of citrate to heparin-PRP decreased aggregation, while addition of heparin to citrate-PRP did not alter aggregation. Aggregation of hirudin-PRP was slightly less than heparin-PRP. Anesthetics affected rat platelet aggregation: the rank order of the maximal extent of ADP-induced aggregation in citrate-PRP was M greater than E = A/K greater than P, and that for AA and collagen in heparin-PRP was E = A/K greater than M = P. The correlation between the effect of the anesthetics and activation of the sympathoadrenal system is discussed. It appeared that of the commonly used anticoagulants and anesthetics, heparin and methoxyflurane had the least influence on rat platelet aggregation.

Compartmentation of 4,6-difluoro-5HT studied by nuclear magnetic resonance in normal and CHS bovine platelets.

A platelet storage pool deficiency (SPD) is present in platelets from cattle with the Chediak-Higashi syndrome (CHS). The most plausible hypothesis for the SPD is that dense granule precursors are simply not formed in CHS megakaryocytes. There is, however, evidence that some recently acquired 5-hydroxytryptamine (5HT) is located in granules and that the granules have an acidic interior. To obtain a greater understanding of the processing of 5HT by SPD platelets, normal and CHS platelets were incubated with 4,6-difluoro-5HT and studied by 19F NMR at 188 mHz. Normal platelets contained 2 compartments for 4,6-difluoro-5HT as indicated by 2 well-developed resonances for each 19F. The resonances were unequal in magnitude. The predominant resonance broadened with lower temperatures and was absent in CHS bovine platelets; it was, therefore, the dense granule compartment. There was only 1 resonance for each 19F in CHS platelets. The chemical shift was identical to the minor resonance, or non-dense granule resonance, found in normal bovine platelets but the resonance width was increased, indicating that some non-dense granule 4,6-difluoro-5HT was in a more restricted environment within CHS platelets than it was in normal platelets.

von Willebrand factor is present in the vascular endothelium from normal dogs and from Doberman pinscher dogs with a plasma von Willebrand factor deficiency.

An immunohistochemical study was undertaken to determine the presence and distribution of von Willebrand factor antigen (vWf:Ag) in blood vessels from normal dogs and from Doberman pinscher dogs with a marked plasma deficiency of vWf. vWf:Ag could not be detected in plasma from the Doberman pinscher dogs by ristocetin- and botrocetin-induced platelet agglutination or by EIA. An ELISA assay revealed vWf:Ag levels that were between 2-4% of that in normal canine plasma. Factor VIII:C activity was 30-46% of normal. The activated partial thromboplastin time (APTT) was increased but not the one-stage prothrombin time (OSPT). Four different antibody preparations were used in this study to detect vWf--a monoclonal and a polyclonal antibody prepared against human vWf and 2 polyclonal antibodies against canine vWf. vWf:Ag was detected with monospecific antibody in endothelial cells in veins, venules, and arterioles from normal dogs and vWf-deficient dogs. The histofluorescence observed in vessels of vWf-deficient dogs was indistinguishable from that observed in vessels from normal dogs.

Effect of exercise, DDAVP, and epinephrine on the factor VIII:C/von Willebrand factor complex in normal dogs and von Willebrand factor deficient Doberman pinscher dogs.

Endothelial cells in biopsied blood vessels from von Willebrand factor (vWf)-deficient Doberman pinscher dogs contain immunologically detectable vWf. These dogs and normal dogs were treated with DDAVP (0.6 microgram/kg) and epinephrine (0.5 microgram/kg/min for 30 minutes) and were exercised, using 5 different exercise protocols, (3-4 m/s for 5-40 minutes at 0-5% grade) to determine if treatments reported to increase plasma factor VIII:C/vWf complex in humans would elevate canine plasma vWf. Following the two most strenuous exercise conditions--30 and 40 minutes--plasma von Willebrand factor antigen (vWf:Ag) increased in normal dogs by 30% and 70%, respectively. Factor VIII:C was increased 47% by the most strenuous exercise conditions. The vWf-deficient dogs would not exercise beyond 30 minutes and neither vWf:Ag nor factor VIII:C activity increased. Following DDAVP, plasma vWf:Ag increased in the normal dogs by 47% and factor VIII:C activity was increased by 48%. Factor VIII:C activity increased by 30% in the vWf-deficient dogs, but there was only a slight change in vWf:Ag. Bleeding time decreased in 5 of 6 vWf-deficient dogs. In the normal dogs vWf:Ag increased by 14% after epinephrine infusion, but factor VIII:C activity did not change; neither parameter was altered in the vWf-deficient dogs. While the factor VIII:C/vWf:Ag complex was increased in the normal dog by exercise and DDAVP, the increase is not as pronounced as has been reported for humans. It is not known whether the poor response of the vWf-deficient dog is due to low levels of vWf in their endothelium or to a release defect.

Canine idiopathic thrombocytopenic purpura.

Canine idiopathic thrombocytopenic purpura (ITP) is a disease in which antibodies bound to the surface of platelets mediate premature platelet destruction by macrophages. ITP in dogs and chronic ITP in humans are analogous diseases. This article draws on information from the literature on ITP in dogs and in humans, and reviews the pathogenesis, diagnosis, and treatment of ITP in dogs.

Evaluation of an additive solution for preservation of canine red blood cells.

The effect of an additive preservative solution on canine red blood cell posttransfusion viability (PTV) and on selected canine red blood cell biochemical parameters was studied. One unit (450 mL) of blood was collected from 6 clinically normal dogs into the anticoagulant citrate phosphate dextrose, centrifuged, and the plasma removed. The red blood cells were then suspended in 100 mL of a saline, adenine, dextrose, and mannitol solution and stored at 4 degrees C. Aliquots were removed for study at 1, 10, 20, 30, 37, and 44 days. The 24-hour PTV of autologous red blood cells was determined using a sodium chromate (51Cr) label. Red blood cell concentrations of 2,3-diphosphoglycerate (2,3-DPG), adenosine-5'-triphosphate (ATP), and pH were also determined. Canine red blood cell PTV, pH, ATP, and 2,3-DPG concentrations decreased during storage (P < .05). The PTV decreased from 94% using day 1 red blood cells to 80% and 75% using day 37 and day 44 red blood cells, respectively (P < .05). Although the mean PTV of the day 44 stored units equaled the Food and Drug Administration (FDA) minimum standard for human red blood cells, the PTV was substandard in 75% of the day 44 units. The FDA standard was exceeded in 83% of the day 37 units. It was concluded that 37-day-old canine red blood cells preserved with a saline, adenine, dextrose, and mannitol solution are of acceptable quality for transfusion.

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability.

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.

Measurement of von Willebrand factor-specific mRNA and release and storage of von Willebrand factor from endothelial cells of dogs with type-I von Willebrand's disease.

OBJECTIVE: To characterize the cellular basis of the plasma von Willebrand factor (vWf) deficiency in Doberman Pinschers with type-1 von Willebrand's disease (vWd). ANIMALS: Five Doberman Pinschers with type-I vWd and 5 clinically normal dogs used as controls. PROCEDURE: Vascular endothelial cell cultures were used to measure constitutive vWf release, thrombin-stimulated vWf release, baseline intracellular vWf concentration, and vWf mRNA expression. RESULTS: Cells cultured from vWd-affected dogs were morphologically indistinguishable from cells cultured from control dogs, but had reductions in constitutive vWf release (6.5-fold) and vWf mRNA content (fivefold) that correlated to the reduction in plasma vWf concentration (sixfold) in these dogs. The 9.0-kb, canine vWf message was identified, using a polymerase chain reaction-amplified segment of the canine vWf gene and was similar in size to the human vWf message. The vWd cells also had reductions in baseline intracellular vWf concentration (15.6-fold) and thrombin-stimulated vWf release (14.5-fold). Additionally, it was observed that normal canine endothelial cells from different anatomic locations were heterogeneous with respect to vWf expression. CONCLUSIONS: These findings suggest that the plasma vWf deficit in dogs with type-I vWd results from decreased endothelial cell production of vWf resulting from either decreased transcription of the vWf gene or abnormalities in mRNA processing/stability. This is similar to findings in human beings with type-I vWd.

Effect of anticoagulant and blood storage time on platelet-bound antibody concentrations in clinically normal dogs.

A solid-phase ELISA to detect antibodies bound to the surface of canine platelets (platelet-bound antibodies) is described. Using this assay, the effect of anticoagulant and storage time of anticoagulant blood on the concentration of antibodies bound to the surface of platelets from clinically normal dogs was investigated. Blood from 3 clinically normal dogs was anticoagulated with acid citrate dextrose, Na3 citrate, and aqueous K3 EDTA and stored on ice for up to 48 hours. Platelet-bound antibody concentration was measured on platelets isolated from anticoagulated blood immediately after venipuncture and subsequent to storage of blood for 24 and 48 hours. Differences in platelet-bound antibody concentrations were investigated among dogs, anticoagulants, and storage times by ANOVA and Bonferroni pair-wise comparison of means. There was no effect of dog on platelet-bound antibody concentration. The effect of time was significant (P < 0.0001), with higher concentration of platelet-bound antibodies detected with increasing storage time. Effect of anticoagulant on platelet-bound antibody concentration was not statistically significant; however, there was a trend to increasing concentration of antibodies bound to platelets isolated from Na3 citrate- and K3 EDTA-anticoagulated blood. Moreover, there was significant (P = 0.02) interaction between anticoagulant and time. Platelet-bound antibody concentration increased with storage of anticoagulated blood prior to platelet isolation and with use of Na3 citrate and K3 EDTA anticoagulants. The preferred anticoagulant for platelet-bound antibody measurement is acid citrate dextrose. Platelet-bound antibody concentration should be determined not longer than 24 hours after blood collection.

Acquisition and aggregation of canine blood platelets: basic mechanisms of function and differences because of breed origin.

A method for obtaining reliable blood platelet yields in canine platelet-rich plasma, using increased sodium citrate concentration, is presented. Maintaining a quiet environment or anesthetizing the animals with thiamylal sodium aids in collection of platelets. Aggregation of platelets from 60 dogs of various breeds in response to arachidonic acid, collagen, adenosine diphosphate, epinephrine, and serotonin was monitored. Canine platelets reversibly or irreversibly aggregated to arachidonic acid. The percentage of arachidonate-irreversible platelets varied from 0% to 100% depending upon the breed of dogs examined. Arachidonate-irreversible platelets also aggregated irreversibly at lower concentrations of collagen and exhibited biphasic irreversible aggregation to adenosine diphosphate and serotonin. Serotonin-induced irreversible aggregation was dependent upon receptor activation and upon arachidonic acid metabolism. Irreversible aggregation to serotonin was associated with release of 3H-serotonin and thromboxane B2 formation, indicating that a couple between the serotonergic receptor and arachidonic acid metabolism may exist.