Competency of blood coagulation in the newborn calf

This study evaluated the haemostatic profiles of a group of 11 female and seven male calves from the day of birth until they were 60 days of age. Similar results were found for both sexes. At birth the plasma activity of the procoagulant proteins, Factors VII, VIII:C, IX, X and XI and fibrinogen were all close to the adult values. Factors VII, VIII:C and fibrinogen increased transiently during the first seven days of life but the increases were not sufficient to influence routine coagulation screening assays such as the activated partial thromboplastin time and the prothrombin time. At birth, the plasma concentration of the protease inhibitor, α₂-macroglobulin, was approximately 50 per cent of adult values and increased slowly during the first seven days of life; the plasma concentration of antithrombin III was higher than that of α₂-macroglobulin. The changes in the plasma concentration of fibronectin paralleled the changes in fibrinogen and Factor VIII:C from birth to 60 days of age; the concentrations of total plasma protein and plasma albumin remained stable and within the adult ranges throughout the 60 days. The plasma concentration of glucose increased transiently during the first 48 hours after birth.     

The effect of administration of a single dose of T-2 toxin on blood coagulation in the rabbit.

The single intravenous administration of T-2 toxin to rabbits at a dosage of 0.5 mg per kg body weight produced an alteration in several blood coagulation parameters. The activities of factors VII, VIII, IX, X and XI were decreased by approximately 40% six hours after T-2 toxin administration. Plasma fibrinogen concentration became elevated within 24 hours after T-2 toxin exposure. Circulating platelet numbers were unaffected by T-2 toxin administration. The similarity of coagulation parameter changes induced by T-2 toxin in animals receiving daily subcutaneous injections of vitamin K (0.5 mg per kg body weight) and those in nonvitamin K supplemented animals suggests that T-2 toxin does not function as a vitamin K antagonist.     

Incidence of Transfusion Reactions and Retention of Procoagulant and Anticoagulant Factor Activities in Equine Plasma

Background: The extent of preservation of clotting factors and incidence of transfusion reactions to noncommercial equine plasma is not documented.
Hypothesis: Equine frozen plasma would retain its coagulation factor activity within the reference range and the incidence of transfusion reactions would be low.
Animals: Ten plasma donor horses. Fifty clinically ill hospitalized horses receiving plasma were reviewed to determine the incidence of reactions.
Methods: In vitro study and retrospective case review. Plasma was prepared by gravity sedimentation from whole blood refrigerated for 48 hours. The activities of factors VII through XII, antithrombin (AT), and Protein C were measured. Factor activities were compared for plasma samples obtained before blood collection (S0), after 48 hours of gravity sedimentation at 5 °C and after plasma separation (S1), and after 90 days of storage at −20 °C (S90). The medical records of 50 consecutive clinically ill horses receiving frozen plasma were reviewed to determine the incidence of transfusion reactions.
Results: The combined effect of plasma harvest, gravity sedimentation, decantation, and freezing caused significant reductions in factors IX, (43% P = .0013), X, (33% P = .0001), XI, (48% P = .0008), AT, (10% P = .02), and Protein C (26% P = .0001). Activities for all factors analyzed, except factor X, remained within the reference ranges. Transfusion reactions were recorded for 5/50 horses.
Conclusions and Clinical Relevance: Clotting factors, AT, and Protein C were well preserved. The incidence of reactions to frozen plasma was 10%.     

Hemostasis development in the lamb fetus and neonate

Fetal and neonatal lamb hemostasis were studied from the 60th day of pregnancy to birth. Platelet counts and blood coagulation, as assessed by tests such as recalcification time and thromboelastography, were similar in fetuses, neonates, and adult sheep. The values of coagulation factors were low, ie, vitamin K-dependent Factors II, VII, IX, and X remained unchanged (30 and 40% of adult reference values) until the last 10 days of gestation, and then increased until birth (40 to 60%). Values of fibrinogen and Factor V followed a similar pattern, although their activities became identical to adult values at birth. Also, we measured values of protein C and antithrombin III, which are synthesized by the liver. The importance of hepatic failure and fetal vitamin K deficiency were discussed. Factors VIII and XII activities increased gradually during pregnancy to reach adult values at birth. Fetal fibrinolytic activity increased. This could not be explained by the values of tissue-type plasminogen activator (it was not detectable) or by the presence of its fast-acting inhibitor, whose concentration did not decrease.     

Stability of hemostatic proteins in canine fresh frozen plasma units

Abstract: The stability of hemostatic proteins, including coagulation factors II, VII, VIII, IX, and X and von Willebrand factor (vWf), in canine fresh frozen plasma (FFP) units stored for up to 1 year was studied. Plasma units from 7 donor dogs were subjected to 4 treatments following collection, including storage at −30°C for 3 months, 6 months, and 1 year and storage at −20°C for 6 months. Coagulant factor activity and vWf concentrations were measured at these times. Significant differences between prestorage and poststorage values were noted for factors VIII, IX, and X, and vWf. Differences seemed to be least pronounced for plasma stored for 3 months; however, a significant interaction between prestorage and poststorage differences and the 4 treatment groups could not be demonstrated. On the basis of factor content in the present study, 15–20 mL/kg of FFP stored for up to 1 year was capable of providing approximately 10–15 U/kg of vWf and factors VIII, IX, X, and II, whereas 10 mL/kg FFP provided at least 10 U/kg of factor VII.     

An overview of hemostasis

The normal hemostatic system is complex yet exquisitely well regulated. Interrelationships exist between responses of the vasculature, circulating platelets, coagulation proteins, and fibrinolytic mechanism. These relationships serve to limit blood loss, preserve tissue perfusion, and stimulate local repair processes. Natural inhibitors of coagulation and fibrinolysis modulate these systems to prevent uncontrolled thrombosis or hemorrhage following pathologic stimuli. Vascular endothelial cells play an important role in the maintenance of a thromboresistant luminal interface with circulating cells and proteins. Normal hemostasis also requires the synthetic, metabolic, and repair processes of the vascular endothelium. The initial vascular response to injury produces brief vasoconstriction and exposes subendothelial substances that attract circulating platelets and activate coagulation proteins. Platelets respond by adherence and aggregation at the site of injury, with subsequent release of substances that mitigate blood loss. Platelet adherence to collagen requires von Willebrand's factor, fibrinogen, fibronectin, and specific glycoprotein receptors on platelet surfaces. Platelet-to-platelet interactions (aggregation) recruit additional platelets to the primary hemostatic plug. Aggregation requires fibrinogen, energy, and calcium. Release of ADP, serotonin, and the contents of intracellular granules as well as generation of prostaglandins prepares platelet surfaces for reactions with the coagulation proteins. Activation of the intrinsic or extrinsic coagulation pathway, or of both, causes formation of fibrin from fibrinogen by means of an elaborate and intricate system that also entraps platelets and activated coagulation proteins. The intrinsic system is activated by the contact of factor XII with a negatively charged surface, most likely collagen. Through a series of reactions with prekallikrein, HMWK, and factors XI, IX, and VIII, the common coagulation pathway is propagated. The extrinsic, or tissue factor, system stimulates both the common pathway and factor IX the intrinsic pathway. Discovery of this stimulation of the intrinsic pathway by factor VII in the extrinsic pathway has stimulated reassessment of the biologic importance of the extrinsic system. The common pathway includes factors X and V and causes thrombin to convert fibrinogen to fibrin. Calcium and platelet phospholipids are substances that have important roles in steps in the coagulation scheme. Once fibrin is formed, factor XIII interacts with the substance, providing a stabilizing effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasma levels of specific coagulation factors and oestrogens in the bitch during pregnancy

During pregnancy in twelve bitches a significant increase was observed in the circulating activity of the specific coagulation factors VII, VIII, IX and XI. Such changes did not occur in two bitches which were bred but did not conceive. Comparison of the plasma procoagulant activity and oestradiol-17 β levels indicated that alterations in the coagulation factor activity may, at least in part, be mediated by the changes in oestrogen levels which occur in the pregnant bitch.     

Effect of citrate concentration on coagulation test results in dogs

OBJECTIVE: To determine the effect of citrate concentration (3.2 vs 3.8%) on coagulation tests in dogs. DESIGN: Original study. ANIMALS: 30 clinically healthy dogs and 12 dogs with hereditary hemostatic disorders. PROCEDURE: Blood was collected from all dogs directly into collection tubes containing 3.2 or 3.8% buffered citrate. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration were measured by use of 3 clot-detection assay systems (2 mechanical and 1 photo-optic). Factor VIII and factor IX coagulant activities (FVIII:C and FIX:C, respectively) were determined by use of a manual tilt-tube method and a mechanical clot-detection device. RESULTS: Significant differences were not detected in median PT, fibrinogen concentration, FVIII:C, or FIX:C between 3.2 and 3.8% citrate for any assay system. A significant prolongation in aPTT for 3.2% citrate, compared with 3.8% citrate, was found in 1 mechanical system. CONCLUSIONS AND CLINICAL RELEVANCE: Citrate concentration does not significantly affect results of most coagulation assays, regardless of assay system. The aPTT was mildly influenced by the citrate concentration, although this was animal-, instrument-, and reagent-dependent. The choice of 3.2 or 3.8% citrate as an anticoagulant for coagulation tests has minimal influence on assay results in healthy dogs or dogs with hereditary hemostatic disorders.     

Comparative stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled fresh frozen plasma

Objective – To evaluate the stability of canine and feline hemostatic proteins in freeze‐thaw‐cycled (FTC) fresh frozen plasma (FFP). Design – Prospective study. Setting – Veterinary Teaching Hospital. Animals – Nine blood donor dogs and 10 blood donor cats. Interventions – Whole blood was collected and separated into packed RBC and plasma units according to standard methods. Each unit of plasma was divided into 2 equal aliquots and frozen (?41°C). One aliquot from each donor (FTC) was then thawed and then refrozen (?41°C) until time of analysis. The second aliquot (nonfreeze‐thaw‐cycled; NFTC) remained frozen until time of analysis. The hemostatic proteins assessed included coagulation factors, anticoagulant factors (antithrombin and Protein C), and adhesive proteins (fibrinogen and von Willebrand Factor). The coagulant activities of factors II, VII, VIII, IX, X, XI, and XII were measured in modified one‐stage activated partial thromboplastin time or prothrombin time assays. Antithrombin and Protein C activities were measured in chromogenic substrate assays. Clottable fibrinogen was measured via the Clauss method, and von Willebrand Factor concentration (vWF:Ag) was measured in an ELISA. A paired t‐test was utilized to identify differences in factor activity or concentration between FTC FFP and NFTC FFP. Measurements and Main results – No clinically or statistically significant differences (all P>0.05) were identified between FTC FFP and NFTC FFP. Conclusions – Refreezing FFP within 1 hour of initial thawing appeared to have no deleterious effects on the hemostatic protein activity or content of that unit. Transfusion of FTC FFP is expected to provide the recipient with comparable replacement of hemostatic proteins as FFP that has remained frozen.     

Stability of canine factor VIII activity and von Willebrand factor antigen concentration in vitro

The in vitro stability of canine factor VIII activity, von Willebrand factor antigen concentration and the ratio of these two factors was studied. Samples were stored for up to 48 hours, either as plasma or as whole blood, at 4 degrees, 20 degrees and 37 degrees C. Factor VIII activity was generally stable in both plasma and whole blood samples for up to 48 hours at 4 degrees or 20 degrees C. The concentration of von Willebrand factor antigen was more stable in samples stored as plasma than whole blood, and for a shorter time than factor VIII activity. Consequently, the stability of the ratio of these two factors was relatively poor in vitro.     

Influence of progesterone and pregnancy on canine fibrinogen values

Progesterone alone or in combination with oestrogen was given to male dogs and nonoestrous bitches by intramuscular injection. Progesterone produced a marked increase in plasma fibrinogen values. The fibrinogen response appeared to be independent of oestrogen administration. No alteration in haematocrit values, platelet count, APTT and OSPT results or the activities of coagulation factors VII, VIII and IX was observed following hormone administration. It is suggested that increased plasma progesterone concentrations may be partly responsible for the two- to three-fold increase in fibrinogen which occurs during gestation in the bitch.     

Coagulation profiles in dogs with congenital portosystemic shunts before and after surgical attenuation

BACKGROUND: Serious postoperative hemorrhage has been reported in dogs after closure of congenital portosystemic shunts (CPS). HYPOTHESIS: In dogs with portosystemic shunting, low coagulation factor activity is responsible for coagulopathy, which can cause complications after surgery. ANIMALS: Thirty-four dogs with CPS and 39 healthy dogs. METHODS: In a prospective study, coagulation times, platelet count, and the activity of 8 coagulation factors were measured in dogs before and after surgical shunt attenuation and in 31 healthy dogs. The effect of abdominal surgery on hemostasis was determined at ovariectomy in 8 healthy dogs. RESULTS: Dogs with CPS had lower platelet counts, lower activity of factors II, V, VII, and X, and increased factor VIII and activated partial thromboplastin time (APTT) compared to healthy dogs. After surgical attenuation, dogs with CPS had decreased platelet counts and activity of factors I, II, V, VII, IX, X, and XI and a prolonged prothrombin time (PT). Ovariectomy resulted in decreased activity of factors VII and X. Six weeks after surgery, portosystemic shunting persisted in 9 of 30 dogs, with no improvement of hemostatic values. CPS dogs without shunting had improved coagulation times and increased activity of factors II, V, VII, and X. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with CPS have lower activity of clotting factors compared to healthy dogs, resulting in a prolonged APTT. Surgical attenuation of the shunt results in increased abnormalities in coagulation times and factors immediately after surgery. Hemostasis is normalized after complete recovery of shunting after attenuation, in contrast to dogs with persistent shunting.     

Disseminated intravascular coagulation in experimentally induced feline infectious peritonitis

Disseminated intravascular coagulation was induced in kittens by intraperitoneal inoculation of feline infectious peritonitis virus (FIPV). Kittens seronegative to FIPV survived significantly (P less than 0.05) longer than those seropositive to FIPV. Pyrexia, anemia, icterus, hyperbilirubinemia, and elevated concentrations of liver-specific enzymes were detected in the inoculated cats. Lesions induced included disseminated fibrinonecrotic and pyogranulomatous inflammation, hepatic necrosis, and widespread phlebitis and thrombosis. Localization of FIP viral antigen and immunoglobulin G was demonstrated in foci of heptic necrosis by immunofluorescence miroscopy. Lymphopenia, thrombocytopenia, hyperfibrinogenemia, and increased quantities of fibrin-fibrinogen degradation products were present in cats after the onset of clinical illness. Depression of factor VII, VIII, IX, X, XI, and XII plasma activities and prolongation of prothrombin and partial thromboplastin times also developed in infected cats. The accelerated onset of clinical disease and mortality in seropositive kittens vs seronegative kittens and the association of virus and antibody in multiple foci of hepatic necrosis suggest an immune-mediated component is involved in the pathogenesis of this disease.     

Coagulation defects of aflatoxin intoxicated rabbits

Twelve New Zealand white rabbits were intoxicated with aflatoxin B1. Most rabbits developed a coagulation defect near the time of death. Immediately prior to death there were significant decreases in factors V, VII, and VIII coagulant activities and fibrinogen concentration without a change in plasma fibrin(ogen) degradation product concentration, platelet number, and detectable plasma fibrin monomers. Microscopic evidence of disseminated intravascular coagulation was present in one rabbit with marked, diffuse hepatic necrosis. Terminal serum albumin concentration was significantly correlated to plasma factors V and VII activities and fibrinogen concentration. The coagulation defect of aflatoxicosis is primarily due to diminished hepatic synthesis of coagulation factors except when hepatic necrosis is severe enough to initiate intravascular coagulation and consumption of coagulation factors.     

Evaluation of the haemostatic profile in the pre- and post parturient mare, with particular focus on the perinatal period.

Various haemostatic analytes were systematically evaluated for four months pre-partum and five months post partum in 14 healthy mares. The plasma fibrinogen concentration and both Factor VIII:C and von Willebrand factor activity showed gradual increases from mid-gestation and reached maximal, or near maximal activity at parturition. These increases were paralleled by an increase in plasma fibronectin concentration, the appearance of fibrinogen degradation products, and a modest rise in antithrombin III concentration. In contrast, the activity of Factor VII and Factor IX, and the one-stage prothrombin (PT) time and the activated partial thromboplastin (APTT) time remained relatively constant throughout the pre- and post parturient period.     

Hemostatic response to surgical neutering via ovariectomy and ovariohysterectomy in dogs

Objective-To investigate the hemostatic response to surgery and compare the response for ovariohysterectomy with that for ovariectomy and to evaluate the usefulness of thromboelastography on plasma samples. Animals-42 female dogs. Procedures-Dogs were assigned to undergo ovariohysterectomy or ovariectomy. Blood samples were collected immediately before and 1, 6, and 24 hours after surgery and stored at -80°C for subsequent analysis. Plasma samples were subjected to thromboelastography after thawing. In addition, coagulation variables were measured, including concentrations of von Willebrand factor antigen, fibrinogen, antithrombin, and protein C; activity of factor VIII; activated partial thromboplastin time; prothrombin time; and thrombin time. The fibrinolytic response was assessed via concentrations of D-dimer, plasminogen, and α-2-antiplasmin (plasmin inhibitor). Results-Substantial hemostatic and fibrinolytic activation was evident after surgery in both groups, as characterized by significantly increased global clot strength and an overall hypercoagulable state at 4 hours after surgery in addition to decreases in von Willebrand factor antigen and factor VIII concentrations and shortened prothrombin and thrombin times. The dogs also typically had activation of the fibrinolytic system, as evidenced by increased postoperative concentrations of D-dimer, plasminogen, and plasmin inhibitor. Differences between the 2 groups could not be detected for any variables. Conclusions and Clinical Relevance-Elective surgery with limited tissue trauma induced hemostatic activation in dogs, which led to hypercoagulability after surgery. A difference between the ovariohysterectomy and ovariectomy groups was not detected. Thromboelastography can be used on plasma samples and may be useful for evaluating patterns over time.     

Effects of exploratory laparotomy on plasma and peritoneal coagulation/fibrinolysis in horses

Plasma and peritoneal fluid samples were collected before and after surgery from 6 horses undergoing a ventral midline exploratory laparotomy and from 6 anesthetized control horses. Coagulation/fibrinolytic components measured in the plasma and peritoneal fluid of these horses included the functional activity of antithrombin III, alpha-2 antiplasmin, plasminogen, and protein C, and the concentrations of fibrinogen and fibrin degradation products. Peritoneal fluid antithrombin III, fibrin degradation products, and plasminogen values were significantly increased after surgery (over time) in principal horses. Compared with control horses, postoperative peritoneal fluid from horses undergoing laparotomy had significantly increased antithrombin-III activity at 12 and 72 hours, alpha-2 antiplasmin activity at 24 hours, fibrin degradation product concentrations at 6, 12, 24, 72, 96, and 144 hours, plasminogen activity at 6, 12, 24, 48, 72 and 96 hours, and protein-C activity at 12, 24, 72, and 96 hours. There were no significant changes in the peritoneal fibrinogen concentration in principal horses. Plasma plasminogen activity was significantly decreased at 24 hours after surgery in principal horses, compared with controls. Changes were minimal in the remaining plasma coagulation/fibrinolytic components of horses undergoing laparotomy. Plasma and peritoneal fluid values of anesthetized control horses did not change.     

Effects of season and sample handling on measurement of plasma alpha-melanocyte-stimulating hormone concentrations in horses and ponies

OBJECTIVE: To investigate effects of sample handling, storage, and collection time and season on plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentration in healthy equids. ANIMALS: 11 healthy Standardbreds and 13 healthy semiferal ponies. PROCEDURE: Plasma alpha-MSH concentration was measured by use of radioimmunoassay. Effects of delayed processing were accessed by comparing alpha-MSH concentrations in plasma immediately separated with that of plasma obtained from blood samples that were stored at 4 degrees C for 8 or 48 hours before plasma was separated. Effects of suboptimal handling were accessed by comparing alpha-MSH concentrations in plasma immediately stored at -80 degrees C with plasma that was stored at 25 degrees C for 24 hours, 4 degrees C for 48 hours or 7 days, and -20 degrees C for 30 days prior to freezing at -80 degrees C. Plasma alpha-MSH concentrations were compared among blood samples collected at 8:00 AM, 12 noon, and 4:00 PM. Plasma alpha-MSH concentrations were compared among blood samples collected in January, March, April, June, September, and November from horses and in September and May from ponies. RESULTS: Storage of blood samples at 4 degrees C for 48 hours before plasma was separated and storage of plasma samples at 4 degrees C for 7 days prior to freezing at -80 degrees C resulted in significant decreases in plasma alpha-MSH concentrations. A significantly greater plasma alpha-MSH concentration was found in September in ponies (11-fold) and horses (2-fold), compared with plasma alpha-MSH concentrations in spring. CONCLUSIONS AND CLINICAL RELEVANCE: Handling and storage conditions minimally affected plasma alpha-MSH concentrations. Seasonal variation in plasma alpha-MSH concentrations must be considered when evaluating pituitary pars intermedia dysfunction in equids.     

Comparison of the coagulation profile of fatty liver haemorrhagic syndrome-susceptible laying hens and normal laying hens

1. The rate of thrombin generation in plasma from Fatty Liver Haemorrhagic Syndrome-susceptible laying hens (FLHS, UCD-003) is more rapid than in plasma from age-matched normal Single Comb White Leghorn (SCWL) laying hens. 2. The rate of thrombin generation in plasma was determined by measuring the biological activity of the specific coagulation proteins, Factors V, VII, VIII, IX and X. 3. The higher activity of Factors V, VII and X in FLHS-susceptible laying hens compared with normal SCWL hens remained consistent after plasma lipid concentrations were reduced. 4. Analysis of the fatty acid composition of plasma phospholipids showed that in normal SCWL laying hens phosphatidylethanolamine contained C18:3n3 whereas it contained C20:3n3 in FLHS-susceptible laying hens. 5. The results suggest that alterations in the composition of the phospholipids that are essential cofactors in the biochemical reactions involved in thrombin generation may be a contributing factor in the development of FLHS.     

Evaluation of EDTA hematology tubes for collection of blood samples for tests of secondary hemostasis in dogs

OBJECTIVE: To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs. ANIMALS: 108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis). PROCEDURES: Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated. RESULTS: Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples. CONCLUSIONS AND CLINICAL RELEVANCE: Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.