Kenneth M. Meyers

An evaluation of the arachidonate pathway of platelets from companion and food-producing animals, mink, and man.

Abstract The arachidonate pathway of human, feline, canine, equine, mink, porcine, and bovine platelets was evaluated by determining the formation of arachidonate-induced malondialdehyde (MDA), thrombin-induced MDA and thrombin-induced thromboxane (Tx) B 2 . In addition, arachidonate-induced platelet aggregation responses were monitored. Arachidonate activated platelets from every animal species evaluated and induced formation of TxB 2 and MDA. There were, however, considerable species differences in the importance of the pathway in mediating the basic platelet reaction. Platelets from mink, pigs, and cows did not aggregate to arachidonate (0.5 mM) and in response to thrombin produced less than 0.5 nmoles of MDA/3 × 10 8 platelets and less than 10 nmoles of TxB 2 /10 11 platelets. Human platelets had a well-developed arachidonate pathway, as they formed more than 1.0 nmoles of MDA/3 × 10 8 platelets and more than 50 nmoles of TxB 2 /10 11 platelets in response to thrombin and irreversibly aggregated in response to arachidonate. Feline platelets exhibited considerable intraspecies variation in the arachidonate pathway. Canine platelets generally formed more than 1.0 nmole of MDA/3 × 10 8 platelets in response to thrombin; yet, platelets from some dogs did not irreversibly aggregate in response to arachidonate. Equine platelets aggregated in response to arachidonate but the aggregation was reversible and they formed between 1.0 and 0.5 nmoles of MDA/3 × 10 8 platelets when incubated with thrombin.

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.

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.

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.

Serotonin involvement in a motor disorder of Scottish terrier dogs

Abstract The effect of altering the concentration of 5-HT or the catecholamines upon an inherited neurological condition of Scottish terrier dogs which is characterized by episodes of muscular hypertonicity was assessed in a blind study. Alpha-methyl-paratyrosine and imipramine did not modify the condition. Amphetamine sulfate induced episodes; however, the episode was generally of shorter duration than the behavioral effect. The severity of the clinical rating was markedly increased by p-CPA. This increased severity was reduced by 5-HTP administration. The peripheral serotonin antagonist xylamidine tosylate did not alter the severity of the disease. Nialamide and 5-HTP had a significant beneficial effect. The increase in severity of the disease which follows a decrease in 5-HT coupled with a decrease in severity with an increase in 5-HT suggest certain serotoninergic neurons are involved in modulation of skeletal muscle tone.

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.

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.

The relationship of serotonin to a motor disorder of scottish terrier dogs

Abstract Serotonin (5-HT) content in brain and whole blood, 5-hydroxy-indoleacetic acid (5-HIAA) concentration in urine and cisternal cerebrospinal fluid (CSF) were determined in normal dogs and Scottish terrier dogs affected with an inherited motor disorder. In addition, the increases in CSF 5-HIAA following probenecid administration were measured in affected and nonaffected dogs at rest, during diethyl ether anesthesia and following p-chloro-phenylalanine administration. There was not a significant difference between the two groups of dogs in any of the mentioned measurements. These data suggest that the primary biochemical defect does not directly involve 5-HT.

Normal and perturbed endothelial cells from canine femoral arteries and femoral veins exhibit heterogeneity in hemostatic properties and growth characteristics

AbstractBackground: We sought to examine the heterogeneity of endothelial cells from the same anatomic site but different vascular systems and described von Willebrand factor (vWF) release and morphological change in response to injury-associated factor in femoral vessels from canine in vitro. Methods: Levels of hemostatic factors (vWF, plasminogen activator inhibitor type 1(PAI-1), antithrombin III (ATIII), in tissue sections and cultured endothelial cells of canine femoral arteries and canine femoral veins were compared by the immunohistochemistry technique. In addition to comparing cell growth density and cell protein contents, cultured femoral arterial endothelial cells (FAECs) and cultured femoral venous endothelial cells (FVECs) were incubated with a series concentration of basic fibroblast factor (bFGF) (1, 10, 100 ng/ml) for up to 48 hours to test the amount of vWF secretion and morphological change. Results: Both in tissue sections and cultured cells, the levels of vWF are higher in FVECs than in FAECs. We were unable to differentiate the level of PAI-1 and ATIII difference between FAECs and FVECs. bFGF (10 ng/ml) significantly increased vWF secretion from cultured FAECs but not from FVECs. The size of cultured FAECs is smaller than of FVECs; however, FAECs have higher amounts of protein contents than FVECs. Conclusions: These comparative studies provide evidence indicating that the characteristics of FVECs differ from those of FAECs. These differences may be indicated heterogeneity with either inherited or acquired thrombotic disease.

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.