Identification of a mutation associated with factor XI deficiency in Holstein cattle.

An autosomal recessive deficiency of blood coagulation factor XI (FXI) has been described in Holstein cattle. Current testing methods are unsuitable for accurately identifying carriers (heterozygotes) of the disease. To identify the molecular basis of this deficiency, a polymerase chain reaction (PCR)-based strategy was implemented to clone and sequence the bovine FXI gene (F11) from animals of different genotypes. Approximately 14 kb of genomic DNA sequence and 1.8 kb of cDNA sequence, corresponding to exon 3 through the 3'-UTR, of the bovine gene were obtained. Comparison of sequences derived from homozygous normal and deficient individuals revealed that FXI deficiency in Holsteins is associated with the insertion of a 76 bp segment [AT(A)(28)TAAAG(A)(26)GGAAATAATAATTCA] within exon 12. This insertion introduces a stop codon that results in a mature FXI protein lacking the functional protease domain encoded by exons 13, 14 and 15. Based on these data, a DNA-based diagnostic test has been developed for accurate genotyping. Using this method, the frequency of the mutated allele has been determined to be 1.2% in a contemporary population of the USA Holstein sires.

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.

Assessment of factor V, VII and X activities, the key coagulant proteins of the tissue factor pathway in poultry plasma.

1. Assay methods were developed for key components of the tissue factor pathway of blood coagulation, namely Factor V, Factor VII and Factor X. Using these assays, plasma from healthy laying hens, cockerels and broilers was shown to contain functional and equivalent amounts of each of these clotting factors. 2. The plasma activities for Factor V, Factor VII and Factor X can only be accurately determined when chicken tissue factor is used to initiate the coagulation mechanism in poultry plasma. Neither human tissue factor nor rabbit tissue factor forms a fully functional enzyme reactive complex with chicken Factor VII. 3. The overall tissue factor pathway coagulation mechanism was evaluated in plasma from laying hens, cockerels and broilers using the one-stage prothrombin time assay. As long as sufficient tissue factor was used, the overall clotting time results obtained with human recombinant tissue factor were not significantly different from those obtained with chicken tissue factor. 4. We conclude that poultry plasma does possess a fully functional tissue factor coagulation mechanism, but homologous chicken tissue factor must be used for in vitro assays of the components of this pathway.

Neutrophil-platelet interactions and their relevance to bovine respiratory disease.

Respiratory disease is a serious and significant health problem for the bovine industry. Classically, the clinical and research focus has been on the putative causative agents and conditions, and their interactions with host inflammatory cells, particularly alveolar macrophages and blood neutrophils. There is, currently, growing acceptance of the concept that blood platelets play a primary role in the inflammatory process. This review explores the implications of such pro-inflammatory activity, especially in the context of neutrophil-platelet interactions, and the species specificity of cellular responses. The relevance of these issues for the treatment and prevention of bovine respiratory disease is also discussed.

Amounts of selected coagulation factors in pre- and post-mortem follicular fluid are similar and do not correlate with molecular mass.

This study was designed to evaluate the amounts of coagulation factors and to determine whether the protein profile in pre-ovulatory ovarian follicular fluid aspirated from ovaries collected from mares at slaughter are representative of that in follicular fluid collected from live animals. The proteins evaluated included, (i) albumin, ceruloplasmin and fibronectin, (ii) the procoagulant plasma proteins, Factor V (FV), Factor VII (FVII), Factor X (FX) and prothrombin, and (iii) the anticoagulant plasma proteins, antithrombin and alpha2-macroglobulin. The amounts of the individual proteins were similar in both types of follicular fluid. There was no correlation between the activity of FV, FVII, FX or prothrombin in follicular fluid and their molecular size although a correlation was found for the other proteins. These results suggest that the procoagulant proteins in follicular fluid are not likely derived from plasma. The total protein content of follicular fluid samples collected from both sources was similar and the results determined with the Biuret, Lowry and Biorad methods were also not significantly different (P>0.05).

Human ovarian follicular fluid has functional systems for the generation and modulation of thrombin.

OBJECTIVE: To determine whether prothrombin is present in follicular fluid and whether the enzymatic pathways for prothrombin activation are similar to those in plasma. DESIGN: Follicular fluid samples collected at the time of oocyte harvest for an assisted reproductive technology procedure (ART) were analyzed for a panel of hemostatic proteins with use of a combination of functional, chromogenic, and Western ligand blot analysis. SETTING: An ART clinic and an academic research laboratory. PATIENT(S): Women undergoing ART. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Determination of components of thrombin generation and thrombin modulatory systems using functional and antigenic assay procedures. RESULT(S): Both prothrombin and components of the prothrombinase enzyme complex, which includes factors V, VII, and X, are present in follicular fluid. Other hemostatic proteins, including factors VIII and IX and vonWillebrand factor, are absent. The direct activation of prothrombin to thrombin is similar in follicular fluid and plasma. Like plasma, inhibitors of both thrombin and thrombin generation, including antithrombin, protein C, and alpha2-macroglobulin, are present in follicular fluid. CONCLUSION(S): Only a select group of hemostatic plasma proteins are present in follicular fluid. There is no direct correlation between molecular size and concentration of individual proteins in follicular fluid. These results indicate that the proteins involved in the thrombin-generating and thrombin modulatory pathways may be derived from ovarian cells, suggesting that thrombin may have a role in folliculogenesis.

Effect of supplementation with dietary seal oil on selected cardiovascular risk factors and hemostatic variables in healthy male subjects.

The average daily consumption of seal oil by the Inuit people is approximately 8-9 g, yet there is very little information on the effect of seal oil consumption on cardiovascular disease risk factors. In this study, 19 healthy, normocholesterolemic subjects consumed 20 g of encapsulated seal oil containing eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3), and docosapentaenoic acid (DPA; 22:5n-3) or 20 g of vegetable oil (control) per day for 42 days. Levels of selected cardiovascular and thrombotic risk factors as well as fatty acid profiles of serum phospholipid and nonesterified fatty acid (NEFA) were determined. EPA levels in serum phospholipid and NEFA increased by 4.3- and 2.7-fold, respectively, in the seal oil supplemented group. DHA levels rose 1.5- and 2.1-fold, respectively, and DPA levels rose 0.5- and 0.7-fold, respectively. Arachidonic acid (AA) levels dropped by 26% in both serum phospholipid and serum NEFA. There was a significant decrease in the ratio of n-6 to n-3 fatty acids in serum phospholipid from 7.2 to 2.1 and a significant increase in the ratio of EPA/AA in NEFA. Ingestion of seal oil raised the coagulant inhibitor, protein C, values by 7% and decreased plasma fibrinogen by 18%. No alterations in other hemostatic variables, including plasma activity of Factors VII, VIII, IX, and X and antithrombin, or in the concentrations of von Willebrand Factor, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, glucose, Apo A-1, or lipoprotein(a) were observed in either group. Other risk factors for cardiovascular disease, including hematocrit, white blood cell count, plasma viscosity, systolic and diastolic blood pressures, heart rate, and platelet aggregation after stimulation with ADP or collagen did not change. Our results indicate that seal oil supplementation in healthy, normocholesterolemic subjects decreased the n-6/n-3 ratio and increased EPA, DHA, and DPA and the ratio of EPA/AA and DHA/AA in the serum phospholipid and NEFA, while exhibiting a modest beneficial effect on fibrinogen and protein C levels.

Factor VII deficiency in a mixed breed dog.

Abnormal bleeding following routine orchectomy of a 5-month-old mixed breed was determined to be due to factor VII deficiency. Although pedigree information was unavailable, failure to respond to vitamin K therapy and the absence of a plasma coagulation inhibitor suggested that the factor VII deficiency was likely inherited rather than acquired.

Docosahexaenoic acid and docosapentanoic acid incorporation into human platelets after 24 and 72 hours: inhibitory effects on platelet reactivity.

Short-term in vitro platelet membrane lipid enrichment studies and feeding trials of human subjects with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown a decreased reactivity in the platelet response to collagen. In this study, exogenous albumin-bound n-3 polyunsaturated fatty acids (PUFAs), namely EPA, DHA and docosapentanoic acid (DPA) were added to platelet suspensions and maintained at 22 degrees C for 24 and 72 hours. Subsequently, the aggregation response to agonist stimulation and the morphological appearance of the platelets were evaluated. A significant enrichment of platelet phospholipids (PL) in n-3 fatty acids occurred upon incubation with n-3 PUFAs in vitro, which was accompanied by a decrease in the aggregation response to collagen and preservation of platelet morphology compared with non-supplemented control platelet preparations. The inhibitory effect of the n-3 PUFAs appeared to be surface mediated in the case of DHA and DPA because the platelet response to agonist returned when the fatty acids were removed by washing. The platelet aggregation response after storage at 22 degrees C was also evaluated in platelet suspensions collected from healthy individuals before and after 42 days of dietary supplementation with seal oil, rich in DPA and DHA. Unlike the in vitro supplementation, in vivo modification and enrichment of platelet PLs by ingestion of seal oil did not appear to improve platelet function during storage relative to the placebo group.

Alterations in blood platelet morphology during aggregate formation in the Asian elephant (Elephas maximus).

The ultrastructure of Asian elephant (Elephas maximus) platelets before and after activation with the agonist platelet activating factor (PAF) was studied. The unactivated platelet has a distinct ultrastructural appearance: the cytoplasm contains large randomly distributed granules but lacks the internal cristae that typify the open canalicular system in many types of mammalian platelets. Following PAF stimulation, large platelet aggregates form, but many platelets remain discrete, with little evidence of pseudopod formation or fusion of membranes. Two types of platelets are visible within the aggregates: those that are morphologically intact and those with gaplike features on the outer membrane and that have become degranulated, appearing as empty swollen sacs. The lack of platelet membrane fusion within the aggregates may permit the reversal of aggregation that is a characteristic response of elephant platelets to PAF.

Bovine platelet adhesion is enhanced by leukotoxin and sialoglycoprotease isolated from Pasteurella haemolytica A1 cultures.

Platelet and fibrin deposits are among characteristic changes observed in lung alveoli of cattle with pasteurellosis induced by Pasteurella haemolytica (biotype A, serotype 1). To determine whether the platelet function could be directly affected by protein products produced by the bacterium, the effects of leukotoxin and O-sialoglycoprotease, culture supernatant antigen secreted by Pasteurella haemolytica A1, on bovine platelet activation were examined by evaluating the enhancement of platelet adhesion to a negatively charged surface relative to untreated control samples. The glycoprotease, or the leukotoxin, was added to plasma free suspensions of bovine platelets and platelet adhesion assessed by two parameters: (i) the number of 3H-adenine-labeled adherent platelets and (ii) the morphology of unlabeled platelets adhering to the charged surface under scanning electron microscopy (SEM). In the presence of calcium, the glycoprotease produced a dose-dependent increase in adhesion. At a concentration of 4.0 micrograms glycoprotease extract protein per 10(7) platelets, a 2-fold increase in adhesion was observed which was similar to the increase in adhesion induced by 0.10 units of thrombin, a known platelet agonist. Both increased platelet adhesion and platelet aggregation were observed with 0.8 microgram glycoprotease extract protein in the presence of calcium. The response of the bovine platelet suspensions to leukotoxin extract protein was dependent on the dosage of the leukotoxin. Adhesion was enhanced at dosages of 25 micrograms leukotoxin protein per 10(7) platelets and below, while at dosages of 50 micrograms and above adhesion was suppressed. Thus, the two proteins secreted by P. haemolytica may interact directly with bovine platelets to initiate platelet aggregation and fibrin formation in alveolar tissue in pneumonic pasteurellosis.

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.

Comparison of in vitro function of neutrophils from cattle deficient in plasma factor XI activity and from normal animals.

Cattle, homozygous for the genetic disorder of factor XI (FXI) deficiency, exhibit less than 2% of normal plasma FXI activity, display an increased bleeding tendency and are more prone to infectious diseases. FXI is one of the protein components of the contact activation system of coagulation that assembles on the surface of circulating neutrophils. Because of the central role of neutrophils in inflammation, the in vitro responses of neutrophils from normal and FXI deficient cattle were compared. Neutrophil degranulation was evaluated by measuring the release of myeloperoxidase and alkaline phosphatase, and the respiratory burst was evaluated by determining superoxide anion production. Neutrophils from FXI deficient animals exhibited a significant increase (P < 0.05) in the spontaneous release of granule contents compared to the cells from normal cattle. Following stimulation with C5a complement derived from normal serum, the neutrophils from the FXI deficient animals exhibited a greater increase (P < 0.05) in both alkaline phosphatase release and superoxide production. In these neutrophils, following stimulation with C3b complement from normal serum, the relative increase in myeloperoxidase release compared to the unstimulated neutrophils was lower than that observed in the neutrophils from normal animals. There was minimal superoxide production in unactivated neutrophils from either normal or FXI deficient cattle and the response to phorbol ester stimulation was similar in both groups of animals. The C5a complement from FXI deficient serum was more effective (P < 0.05) in stimulating alkaline phosphatase release and superoxide production in normal neutrophils than the equivalent fraction from FXI deficient serum while the C3b complement from the FXI deficient serum was less effective than the normal serum fraction at inducing myeloperoxidase release from normal neutrophils. The results indicate that the differences in the in vitro neutrophil function are likely related to a variation in the function of the contact activation system on the neutrophil surface between normal and FXI deficient animals.

Oncotic, hemodilutional, and hemostatic effects of isotonic saline and hydroxyethyl starch solutions in clinically normal ponies.

OBJECTIVE: To evaluate the oncotic, hemodilutional, and hemostatic effects of IV infusions of a large volume of isotonic saline solution and 2 doses of 6% hydroxyethyl starch (HES) in clinically normal ponies. ANIMALS: 12 adult ponies. PROCEDURE: Ponies were assigned to 3 treatment groups and received the following IV infusions: 80 ml of 0.9% sodium chloride/kg; 10 ml of 6% HES (in 0.9% sodium chloride)/kg; or 20 ml of 6% HES (in 0.9% sodium chloride)/kg. Blood samples were collected for determination of colloid oncotic pressure (COP), PCV, plasma total protein concentration, platelet count, von Willebrand factor antigen (vWf:Ag) activity, fibrinogen concentration, prothrombin time, activated partial thromboplastin time (APTT), and factor VIII coagulant (FVIII:C) activity. A rocket immunoelectrophoretic procedure was used for determination of vWf:Ag activity. A modification of the APTT assay was used for determination of FVIII:C activity. Cutaneous bleeding time was determined, using a template method. RESULTS: Mean COP was persistently increased over baseline values in the face of hemodilution in HES-treated ponies. Prothrombin time, APTT, and fibrinogen concentrations decreased after infusions and vWf:Ag and FVIII:C activities were decreased in dose-dependent manner in HES-treated ponies. Though cutaneous bleeding time was not significantly affected in ponies of any group, a trend toward prolongation of bleeding time was evident in ponies receiving 20 ml of HES/kg. This trend appeared to be associated with marked decrement in vWf:Ag activity at this dosage. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of HES in clinically normal ponies increases COP, and exerts dose-dependent hemodilutional effects and dose-dependent effects on specific hemostatic variables. Thus, HES may be useful for resuscitative fluid treatment of horses.

An inherited platelet function defect in a Simmental crossbred herd.

An inherited bleeding disorder, resembling Simmental hereditary thrombopathy (SHT), has been identified in a Simmental crossbred herd. In an affected bull calf, initially evaluated because of excessive bleeding from a vaccination site, the platelet aggregation response to the agonist, adenosine-diphosphate (ADP) was essentially absent and the aggregation response to platelet activating factor (PAF16) was reduced by at least 70%. The initial laboratory assessment of platelet function in the dam and sire yielded results which were within normal limits. The sire was not available for further testing. The dam, also a daughter of this sire, was subsequently shown to have a partially reduced aggregation response to ADP. Of 18 other offspring of the sire evaluated, 6 were also identified as having a partially impaired aggregation response. The maximum aggregation response to ADP and PAF16 in these 6 calves was approximately 50% of the level exhibited by unaffected animals. In contrast, the coagulation profiles were normal for all animals except for a heifer calf which also exhibited a partially impaired aggregation response. The plasma level of the coagulation protein, factor XI, was reduced in this heifer calf which suffered a fatal hemorrhage following dehorning. This report appears to be the 1st to have identified animals putatively heterozygous for SHT on the basis of the in vitro platelet aggregation response to ADP.

Bovine erythrocyte haemolysates enhance plasminogen activation by tissue-type plasminogen activator.

An active fraction that accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) was purified from a haemolysate of bovine erythrocytes. When the haemolysate was mixed with t-PA, it produced a 2- to 3-fold increase in plasminogen activation as measured by an insoluble fibrinolytic assay system and a soluble amidolytic assay system with the chromogenic substrate S-2251. Zymographic analysis showed that, while the haemolysate increased t-PA activity, it did not alter the electrophoretic characteristics of the t-PA nor did it induce any fibrinolysis in the absence of t-PA or plasminogen. The haemolysate was devoid of plasmin and plasminogen activator activity but was most effective in accelerating plasminogen activation by t-PA in the presence of substrate. Based on the purification characteristics of the active fraction in the haemolysate, it appears to have a molecular weight of less than 10 kDa.

Fibronectin concentrations correlate with ovarian follicular size and estradiol values in equine follicular fluid.

The amounts of total protein, albumin, fibronectin, alpha 2-macroglobulin (alpha 2-M), immunoglobulin G, ceruloplasmin and antithrombin were determined in fluids collected from 53 preovulatory equine follicles and compared with the contents of estradiol-17 beta, progesterone and androstenedione, with follicle size and the amounts of the equivalent proteins in normal equine plasma. The concentration of fibronectin and the fibronectin/albumin ratios increased significantly with follicle size and with follicular estradiol levels. The alpha 2-M levels and alpha 2-M/albumin ratios correlated with follicle size but not with hormone content. Both fibronectin and alpha 2-M were present in lower amounts in follicular fluid compared with plasma while the other proteins were present in similar amounts. Among the proteins evaluated, there was a positive correlation between the amount of the protein in the follicular fluid and the molecular weight of the protein.

Modulation of bovine platelet function by C-reactive protein.

The addition of C-reactive protein (CRP) to bovine platelets suspended in homologous plasma consistently produced a reversible aggregation response following stimulation with either platelet activating factor or adenosine diphosphate while untreated control samples exhibited irreversible aggregation. This deaggregation response was independent of the amount of CRP incorporated into the platelet aggregates but did appear to be mediated through a component either present in bovine plasma or loosely bound to the exterior platelet membrane. The aggregation response of bovine platelets, separated from plasma by gel-filtration, was not affected by the addition of CRP to the platelet suspensions. It is proposed that one of the physiological actions of bovine CRP is to modulate platelet function.