Whole blood platelet aggregation in uremic dogs

Whole blood platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate were determined by use of the impedance method in 22 dogs with serum urea concentrations greater than or equal to 20 mmol/L, which was attributable to renal disease, and in 25 healthy control dogs. The median changes in impedance for the control dogs were 23 ohms for collagen, 18 ohms for arachidonic acid, and 6 ohms for adenosine diphosphate. The median changes in impedance in uremic dogs were 25 ohms for collagen, 21 ohms for arachidonic acid, and 15 ohms for adenosine diphosphate. There were no significant differences in platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate between uremic and control dogs. Hemorrhagic tendencies were not detected in uremic dogs by use of whole blood platelet aggregation. Results of this study suggest that platelet aggregation by use of the whole blood platelet aggregometer is not abnormal in uremic dogs, but does not exclude the possibility of a platelet aggregation defect undetected by the whole blood system.     

Whole blood platelet aggregation in dogs with liver disease

Whole blood platelet aggregation was determined in response to collagen, arachidonic acid, and adenosine diphosphate in 20 dogs with liver disease and in 20 control dogs. Platelet aggregation in response to collagen and arachidonic acid was reduced in dogs with liver disease, compared with control dogs (P less than 0.05), whereas there was no significant difference in platelet response to adenosine diphosphate between the 2 groups of dogs. Adenosine diphosphate was found not to be a reliable aggregation agent for determination of whole blood platelet aggregation in dogs. Dogs whose platelets did not aggregate in response to collagen and/or arachidonic acid manifested bleeding tendencies that could be attributed to platelet dysfunction.     

Characterization of the equine platelet aggregation response.

Equine platelet aggregation responses to bovine collagen, adenosine diphosphate (ADP), serotonin, epinephrine, and arachidonate in a platelet aggregometer were recorded. Equine platelets exhibited irreversible aggregation when incubated with ADP at a final concentration of 10 microM and bovine collagen. A secondary aggregation wave was recorded from platelets from certain horses at final ADP concentrations of 1 to 5 microM. Serotonin and arachidonate induced a weak reversible aggregation response, but a response was not observed following epinephrine addition. Equine platelet aggregation was influenced by concentration of anticoagulant (sodium citrate). Platelet aggregation responses at 37 C were indistinguishable from those recorded at 39 C. Platelet aggregation responses also were altered if the aggregation tests were not performed within 4 hours of blood sample acquisition. An assessment of platelet aggregation from multiple blood samples from the same horse indicated that the procedures described provide a reliable method to assess equine platelet aggregation in vitro.     

Effects of low-dose aspirin and specific thromboxane synthetase inhibition on whole blood platelet aggregation and adenosine triphosphate secretion in healthy dogs.

Platelet aggregation and adenosine triphosphate (ATP) secretion in response to arachidonic acid (10 microM) or collagen (5 micrograms/ml) were compared in healthy, adult female Beagles treated with low-dosage aspirin (3.5 mg/kg of body weight, PO, q 12 h for 7 treatments) or with CGS 12970, a specific thromboxane synthetase inhibitor (10 mg/kg, PO, q 8 h for 10 treatments). Platelet aggregation was assessed in whole blood by use of an electrical impedance method. Baseline values obtained prior to treatment served as controls. Addition of arachidonic acid to blood from nontreated dogs resulted in significantly (P less than 0.001) increased impedance, but had no effect in blood from dogs treated with either aspirin or CGS 12970. Treatment with CGS 12970 or aspirin significantly (P less than 0.001) decreased platelet ATP secretion in response to arachidonic acid, compared with baseline values; however ATP secretion in aspirin-treated dogs was significantly (P less than 0.01) less than ATP secretion in CGS 12970-treated dogs. Differences in platelet aggregation were not observed between control dogs and aspirin- or CGS 12970-treated dogs in response to collagen as an aggregant, however, collagen-induced platelet ATP secretion was significantly (P less than 0.001) decreased in dogs treated with aspirin, compared with control values and values from dogs treated with CGS 12970. In dogs treated orally with 0.1, 0.2, 1.0, or 10 mg of CGS 12970/kg, dose-dependent inhibition of arachidonic acid-induced platelet aggregation was observed, with impedance changes not observed at the 10-mg/kg dosage and normal platelet aggregation associated with the 0.1-mg/kg dosage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of platelet aggregation in ovine blood using a new impedance aggregometer

Background: Whole blood platelet aggregometry (impedance) is an important method to investigate platelet function disorders. Examination of hemostatic function in sheep is important with respect to their role as an animal model of human disease. Objective: The aim of this study was to evaluate and optimize selected methodological aspects (anticoagulant, agonist concentration) of impedance aggregometry in ovine blood using the new Multiplate 5.0 analyzer. Methods: Blood samples were collected in hirudin anticoagulant from 40 clinically healthy sheep. Samples from selected sheep were collected in citrate, with or without the addition of calcium chloride. The agonists adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid, and thrombin receptor‐activating peptide (TRAP) were added in several concentrations to induce aggregation. Results: Based on maximum aggregation values and internal precision, no significant difference was found between ADP concentrations of 3–10 μmol/L and collagen concentrations of 3–5 μg/mL (P>.05). The lowest interindividual variation of approximately 3–4‐fold was seen with 4 and 5 μmol/L ADP and 4 and 5 μg/mL collagen. Ristocetin, arachidonic acid, and TRAP did not induce significant aggregation at any concentration. Aggregation results were significantly lower when measured in citrate‐ vs hirudin‐anticoagulated blood, regardless of the presence of calcium chloride. Conclusions: Our results indicate that the multiplate impedance aggregometer is suitable for the measurement of platelet aggregation in sheep using optimal agonist concentrations of 4–5 μmol/L ADP and 4–5 μg/mL collagen. Hirudin‐anticoagulated blood is the preferred sample material.     

Platelet Hyperfunction in Dogs With Malignancies

In vitro platelet aggregometry was performed on whole blood samples from 59 dogs with malignancies and 24 control dogs. Three reagents were used for the aggregation studies: collagen, arachidonic acid, and adenosine diphos-phate (ADP). The parameters measured to evaluate response to collagen included delay in the aggregation response, slope of the aggregation curve, maximum aggregation, and adenosine triphosphate (ATP) secretion. The platelets of dogs with malignancies exhibited significantly ( P < .05) shorter delays in the aggregation response, higher maximum aggregation, and higher ATP secretion when compared to control dogs. For the weaker reagents, ADP and arachidonic acid, the lowest concentration resulting in aggregation was determined. Platelets of dogs with malignancies tended to aggregate in response to lower concentrations of ADP than did those of controls ( P < .05). The response of platelets to the concentrations of arachidonic acid employed in this study was poor, with few samples achieving measurable aggregation. The findings of this study suggest that dogs with malignancies have hyperaggregable platelets.     

Preliminary studies of a platelet function disorder in Simmental cattle.

A bleeding disorder due to abnormal platelet function occurs in Simmental cattle. Whole blood from these animals underwent good clot retraction. Platelet aggregation in response to adenosine diphosphate (ADP) and collagen in a whole blood aggregation system was markedly impaired. Normal bovine platelets in a whole blood aggregation system showed very little aggregation in response to epinephrine and arachidonic acid. Aggregation in platelet-rich plasma was negligible in response to ADP, collagen and thrombin. Dense granule release of radiolabelled serotonin from the platelets of one affected cow was similar to that of normal bovine platelets. Platelet membrane glycoprotein electrophoresis with the platelets of one affected cow revealed no quantitative abnormalities. These findings reveal similarities and differences in thrombopathic Simmental platelet function when compared to human Glanzmann's thrombasthenia and Basset Hound thrombopathia.     

Studies on platelet aggregation using the Born method in normal and uraemic dogs

Using the Born method, based on light transmission in platelet rich plasma, the minimum effective concentration (threshold values) of several platelet agonists for inducing maximum platelet aggregation was determined in healthy dogs. The final concentrations of aggregation agonists were as follows: adenosine diphosphate (ADP) (0.5-50 micromol/L; n = 75 healthy dogs), collagen (0.5-20 mg/mL; n = 75), thrombin (0.1-5 IU/mL; n = 75), ristocetin (1-10 mg/mL; n = 10), and epinephrine (5-50 micromol/L; n = 10). Reference values for maximum aggregation with a lower limit of > 80% were achieved for agonist concentrations 25 micromol/L ADP (80-98%), > or = 10 microg/mL collagen (80-96%), and > or = 1 IU/mL thrombin (80-97%). None of the concentrations of epinephrine and ristocetin used in this study induced quantitative aggregation in the whole group of healthy dogs. We also studied platelet aggregation in 14 uraemic dogs using selected concentrations of aggregation agonists. Aggregation was significantly decreased in uraemic dogs using intermediate agonist concentrations, i.e., in the region of the threshold concentration. In contrast, maximum aggregation was increased in uraemic patients compared to reference values using low concentrations of all three agonists (ADP: 1 micromol/L, collagen: 1 microg/mL, and thrombin: 0.1, 0.2 IU/mL).     

Vincristine impairs platelet aggregation in dogs with lymphoma

Platelet aggregation before and after administration of 0.5 mg/m2 of vincristine (VCR) was evaluated in 7 dogs with spontaneously occuring lymphoma. Aggregation on platelet-rich plasma separated from blood collected in 3.8% sodium citrate was performed using adenosine diphosphate (ADP), arachidonic acid (AA), and collagen (COL) as agonists. The slope for aggregation in response to ADP was significantly lower after administration of VCR (P = .032). Maximal aggregation after administration of VCR was significantly lower in response to ADP, COL, and AA (P = .03, P = .04, and P = .03, respectively).     

Phenylbutazone inhibition of equine platelet function.

Nonsteroidal anti-inflammatory drugs impair platelet aggregation and secretion in man, pigs, and rabbits and inhibit platelet thromboxane/prostaglandin synthesis. The present investigation studied the effects of phenylbutazone on platelet aggregation and bleeding times in the horse. Aggregation responses to adenosine diphosphate and collagen were markedly impaired 15 minutes and 2 hours after treatment, but 4 hours after treatment, platelet responses approximated those prior to treatment. The in vivo effect of phenylbutazone correlated with its plasma concentrations. Phenylbutazone, like aspirin, appeared to exert its effect by inhibiting thromboxane/prostaglandin synthesis, because thrombin-induced malondialdehyde formation was inhibited. However, unlike aspirin, free arachidonate-induced malondialdehyde synthesis was reduced but not eliminated, which suggested that phenylbutazone may have more than one site of action. Although collagen-induced platelet aggregation was impaired, a response was still present, and bleeding times were not altered by phenylbutazone treatment. To account for these findings, it is proposed that equine platelets can respond to collagen by thromboxane/prostaglandin independent pathways. The physiologic and pathophysiologic importance of these findings is discussed.     

Platelet aggregation in dogs after sedation with acepromazine and atropine and during subsequent general anesthesia and surgery.

Platelet aggregation and adenosine triphosphate (ATP) release were measured by use of the impedance method in blood samples obtained from 25 adult female Beagles before and after sedation with acepromazine (0.13 mg/kg of body weight) and atropine (0.05 mg/kg), and during general anesthesia. General anesthesia was induced by IV administration of thiamylal (average dosage, 2.1 mg/kg; range, 1.2 to 4.2 mg/kg) and was maintained with halothane in oxygen. Samples of jugular venous blood were obtained from each dog, using citrate as anticoagulant. Platelet count was done on each sample. Platelet aggregation and ATP released from the aggregating platelets were measured within 2.5 hours of sample collection, using a whole-blood aggregometer. Adenosine diphosphate (ADP) or collagen was used as aggregating agent. For each aggregating agent, platelet aggregation and ATP release were measured over 6 minutes. After sedation with acepromazine and atropine, significant (P < 0.01) reduction was observed in platelet count (from median values of 341,000 cells/microliters to 283,000 cells/microliters) and in the ability of platelets to aggregate in response to ADP (from 14.0 to 7.0 omega). During the same period, maximal release of ATP in response to collagen also was reduced (from 5.56 mumol to 4.57 mumol; P < 0.01); however, this difference ceased to be significant when ATP release was normalized for platelet count. During general anesthesia and surgery (200 minutes after sedation), platelet count and aggregation responses to ADP and collagen had returned to presedation values. None of the dogs in this study appeared to have hemostasis problems during surgery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Failure of aspirin to impair bovine platelet function

The effect of aspirin on bovine platelet function and thromboxane A2 (TXA2) production in stimulated platelets was evaluated. A single dose of aspirin (100 mg/kg of body weight) was administered orally to Holstein cows, and blood samples were obtained before and at regular intervals for 7 days after treatment. The production of TXA2 was assessed by measuring the stable metabolite thromboxane B2, using a specific radioimmunoassay. Within 4 hours of aspirin administration, the production of TXA2 was significantly (P less than 0.05) decreased, irrespective of whether collagen, adenosine diphosphate, or platelet activating factor was used to initiate platelet aggregation. Despite the inhibition of TXA2 release from the stimulated platelets, platelet function, assessed by initial rate of aggregate formation and extent of aggregation, was unaffected by aspirin administration. The extent of aggregate formation in response to collagen, adenosine diphosphate, or platelet activating factor was independent of the amount of TXA2 released from platelets before and after aspirin treatment. The results suggested that TXA2 formation is not the primary biochemical pathway involved in the aggregation of stimulated bovine platelets.     

Platelet aggregation studies in acute experimental canine ehrlichiosis

BACKGROUND: Thrombocytopenia is the most common and consistent hematologic finding in patients with canine monocytic ehrlichiosis. Dogs that recover from the severe thrombocytopenia still show bleeding tendencies, which suggest that platelet dysfunction is present. OBJECTIVES: The purpose of this study was to determine the occurrence and duration of platelet dysfunction in dogs with ehrlichiosis and to assess whether dysfunction is related to thrombocytopenia. METHODS: Ten adult male and female mongrel dogs were used in the study; 7 were inoculated intravenously with whole blood containing Ehrlichia canis, and 3 were used as controls. Platelet aggregation (with collagen/epinephrine and adenosine diphosphate (ADP)/epinephrine) and platelet counts were evaluated weekly for 112 days. RESULTS: The infected group showed a decrease in platelet aggregation response to collagen/epinephrine and ADP/epinephrine on days 7, 14, 21, 28, and 35 (P <.05). Thrombocytopenia was observed in all infected animals from day 7 to 35 postinfection (P <.05). CONCLUSIONS: The tendency of dogs infected with E canis to bleed may be related not only to thrombocytopenia but also to platelet dysfunction associated with the disease.     

Platelet aggregation in platelet suspensions from Mongolian gerbils (Meriones unguiculatus).

Blood obtained from Mongolian gerbils (Meriones unguiculatus) by cardiac puncture was used to develop a method for preparing platelet suspensions suitable for biochemical and aggregometric studies. The aggregation responses of gerbil platelet suspensions to 10 microM adenosine diphosphate and to 0.2 U/mL thrombin were immediate and irreversible. In addition, thrombin produced a short lag period. Bovine acid-soluble collagen (1:800) produced a long lag period coupled with an irreversible aggregation. Indomethacin (10 microM) significantly depressed adenosine diphosphate--induced aggregation but had no effect on thrombin-induced aggregation. Indomethacin significantly prolonged the lag period and reduced the rate of collagen-induced aggregation. Our results strongly indicate that platelet suspensions from the Mongolian gerbil, an animal which is responsive to hypercholesterolemic diets, may be useful in studying dietary lipid factors which influence platelet aggregation.     

Comparative Effects of Phenylbutazone, Naproxen and Flunixin Meglumine on Equine Platelet Aggregation and Platelet Factor 3 Availability in vitro

Nonsteroidal anti-inflammatory drugs are commonly used in the treatment of inflammatory conditions, and have potential value in the treatment of thrombotic disease in the horse. This study compares the potency of three nonsteroidal anti-inflammatory drugs phenylbutazone, naproxen (equiproxen) and flunixin meglumine (banamine) with respect to their effects on equine platelets. Two functional responses of horse platelets were evaluated in vitro: their ability to aggregate and their ability to make available platelet factor 3 procoagulant activity.

Flunixin at a concentration of 10^-6 M significantly depressed the maximum degree of adenosine diphosphate-induced (10^-6M) aggregation while much higher concentrations of phenylbutazone and naproxen (5 X 10⁵M) were required to produce similar effects. None of the non-steriodal anti-inflammatory drugs significantly affected the duration of the lag phase or the initial velocity of adenosine diphosphate-induced aggregation within the range of drug concentrations used (10^-6-10^-3M). The lag phase and initial velocity of acid-soluble collagen-induced aggregation were significantly affected by 10^-6 M flunixin and 10^-4 M phenylbutazone or naproxen was required to produce equivalent effects. Concentrations of 5 X 10^-6 M flunixin and 5 X 10^-4 M phenylbutazone or naproxen were required to significantly depress the degree of collaen-induced aggregation of horse platelets.

Although the effects of the nonsteroidal anti-inflammatory drugs were qualitatively similar, flunixin was a much more potent inhibitor of platelet aggregation than either of the other two drugs (which were equipotent). At very high drug concentrations (5 X 10^-4 M and greater), all three drugs produced the same degree of inhibition of equine platelet aggregation.

Platelet factor 3 activity was made available by exposing horse platelets to 10^-5 M adenosine diphosphate or 1:800 acid-soluble collagen; but not by exposure to a suspension of kaolin particles. Only a small portion of the total platelet factor 3 activity was made available on stimulation with either adenosine diphosphate or collagen. Pretreatment of horse platelets with any of the nonsteroidal anti-inflammatory drugs (10^-4 M concentration) had no significant effect on adenosine diphosphate or collagen-induced platelet factor 3 availability.

     

Platelet aggregation studies in dogs with acute Ehrlichia platys infection

Ten adult dogs (5 Beagles and 5 mixed-breed dogs) were inoculated IV with canine platelets containing Ehrlichia platys. Inclusions and morulae of E platys developed in platelets of infected dogs at 10 to 14 days after inoculation, followed by marked thrombocytopenia at 14 to 21 days. Parasitemia and marked thrombocytopenia recurred at 24 to 28 days after inoculation. Increased numbers of megakaryocytes were observed in marrow aspirate smears from infected dogs, indicative of regenerative thrombocytopenia. Prior to infection, platelet-rich plasma from these dogs was determined to have similar aggregatory response to arachidonate. After infection with E platys, the aggregatory response of platelet-rich plasma to collagen or 3 dilutions of adenosine diphosphate was evaluated. A statistically significant (P less than 0.05) inhibition of platelet aggregatory response to the lowest dilution of adenosine diphosphate was detected for mixed-breed dogs, whereas aggregation responses were unchanged in Beagles. Results indicate that platelet activation may occur in dogs with acute ehrlichial infection.     

Identification of an Intrinsic Platelet Function Defect in Spitz Dogs

A recently identified intrinsic platelet function defect in 2 Spitz dogs is described. Both affected dogs had a history of chronic intermittent bleeding primarily from the nasal, oral, and gastrointestinal mucosa. Platelet aggregation in response to adenosine diphosphate (ADP), collagen, and platelet activating factor (PAF) was absent; however, platelet shape change did occur. Platelets aggregated in response to gamma thrombin, although a delayed onset and a reduced velocity of aggregation were present. Platelet ¹⁴C-serotonin release was diminished in response to collagen and PAF. Glycoprotein Illa was detected on the surface of platelets by flow cytometry. Platelets were morphologically normal under light and electron microscopy. Two male Spitz dogs, related to one of the affected dogs, did not have a bleeding diathesis. Collagen-induced platelet aggregation, however, was diminished in these 2 dogs. This platelet defect most closely resembles the defect described in Basset hounds.     

Effects of aspirin and propranolol alone and in combination on hemostatic determinants in the healthy cat

The effects of aspirin (75 mg orally/average-size cat) and propranolol (5 mg orally every 8 hours/average-size cat) alone and in combination on hemostatic determinants in healthy cats were studied. In cats, aspirin alone did not cause a significant effect in platelet numbers, plasma fibrinogen, activated partial thromboblastin time, prothrombin time, thrombin time, or platelet aggregation response to adenosine diphosphate. Aspirin did, however, significantly reduce the degree of aggregation induced by acid soluble collagen. Propranolol alone or in combination with aspirin did not cause a significant effect on platelet numbers, plasma fibrinogen, activated partial thromboblastin time, prothrombin time, thrombin time, or platelet aggregation in response to acid soluble collagen, adenosine diphosphate, or adrenaline. It was concluded that aspirin alone at the recommended dosage of one-quarter of a 5-grain tablet (1.25 grains or 75 mg) every other day will significantly affect platelet function and may be of value in the prevention of thromboembolic disease in the cat.     

Platelet Dysfunction Associated With Immune-Mediated Thrombocytopenia in Dogs

The effect of antiplatelet antibody on in vitro platelet function was investigated in 15 dogs with immune-mediated thrombocytopenia (ITP). Platelet aggregation was assessed after addition of serum from healthy dogs (n = 5) or dogs with ITP (n = 15) to platelet-rich plasma from a healthy donor dog. The aggregation responses to adenosine diphosphate, thrombin, and collagen/epinephrine were measured as the maximum aggregation observed after 2 minutes. In 13 of 15 dogs with ITP, maximal aggregation was significantly inhibited in response to ADP, thrombin, or collagen/epinephrine. The slope of the aggregation curve was decreased after addition of serum from 9 of 15 patients. A polyclonal rabbit anti—dog platelet antiserum induced inhibition of aggregation with all 3 agonists.
Serum from control dogs neither inhibited nor activated platelet aggregation. Aggregation experiments were repeated with all 3 agonists after addition of patient immunoglobulin (lg)G or IgG from a healthy dog to platelet-rich plasma. The IgG fraction from 9 of 10 dogs with ITP suppressed platelet aggregation. The IgG fraction from polyclonal rabbit anti—dog platelet antiserum inhibited platelet aggregation with all agonists. These results suggest that many canine ITP patients have circulating antibodies that, in addition to causing platelet destruction, may cause platelet dysfunction.     

Effects of hydrocortisone and aminophylline on the aggregation of equine platelets in vitro

The purpose of this study was to evaluate in vitro the effects of hydrocortisone and aminophylline on adenosine diphosphate (ADP)-induced platelet aggregation in horses. Blood samples from 30 healthy Thoroughbred horses were collected by via jugular venipuncture to assess platelet aggregation. Platelet-rich and platelet-poor plasma were prepared from all samples by centrifugation and divided into three different aliquots. In the first aliquot, platelet aggregation was measured after platelet activation with 1 µM and 0.5 µM ADP (Group A). In the other two aliquots, the effect of a 10 min preincubation with hydrocortisone (Group B) or aminophylline (Group C) on ADP-induced aggregation at final ADP concentrations of 1 µM and 0.5 µM was observed. Platelet aggregation, recorded by an aggregometer, was evaluated by measuring the maximum degree of platelet aggregation and the initial velocities of platelet aggregation were obtained. Our results demonstrated the inhibitory effect of hydrocortisone and the induction effect of aminophylline on equine platelet responses in vitro.