Modelling thrombin generation in human ovarian follicular fluid.

A mathematical model is constructed to study thrombin production in human ovarian follicular fluid. The model results show that the amount of thrombin that can be produced in ovarian follicular fluid is much lower than that in blood plasma, failing to reach the level required for fibrin formation, and thereby supporting the hypothesis that in follicular fluid thrombin functions to initiate cellular activities via intracellular signalling receptors. It is also concluded that the absence of the amplification pathway to thrombin production in follicular fluid is a major factor in restricting the amount of thrombin that can be produced. Titration of the initial concentrations of the various reactants in the model lead to predictions for the amount of tissue factor and phospholipid that is required to maintain thrombin production in the follicle, as well as to the conclusion that tissue factor pathway inhibitor has little effect on the time that thrombin generation is sustained. Numerical experiments to determine the effect of factor V, which is at a much reduced level in follicular fluid compared to plasma, and thrombomodulin, illustrate the importance for further experimental work to determine values for several parameters that have yet to be reported in the literature.

Effects of doxycycline, amoxicillin, cephalexin, and enrofloxacin on hemostasis in healthy dogs.

OBJECTIVE: To determine the effects of enteral administration of doxycycline, amoxicillin, cephalexin, and enrofloxacin at therapeutic dosages for a typical duration on hemostatic variables in healthy dogs. ANIMALS: 14 Beagles. PROCEDURE: Doxycycline (10 mg/kg, PO, q 12 h), amoxicillin (30 mg/kg, PO, q 12 h), cephalexin (30 mg/kg, PO, q 12 h), and enrofloxacin (20 mg/kg, PO, q 24 h) were administered in random order to 10 healthy dogs at standard therapeutic dosages for 7 days, with a 7-day washout period between subsequent antimicrobials. In addition, 4 Beagles served as control dogs. Variables were evaluated before and after antimicrobial administration; they included platelet count, Hct, 1-stage prothrombin time (PT), activated partial thromboplastin time (PTT), fibrinogen concentration, and platelet function. Platelet function was assessed via buccal mucosal bleeding time, aggregation, and a platelet-function analyzer. RESULTS: Administration of all antimicrobials caused a slight prolongation of 1-stage PT and activated PTT and slight decrease in fibrinogen concentration. Cephalexin caused a significant increase in 1-stage PT and activated PTT, amoxicillin caused a significant increase in activated PTT, and enrofloxacin caused a significant decrease in fibrinogen concentration. Platelet count or function did not differ significantly after administration of any antimicrobial. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of commonly used antimicrobials in healthy dogs resulted in minor secondary hemostatic abnormalities, with no change in platelet count or function. Although these changes were clinically irrelevant in healthy dogs, additional studies of the effects of antimicrobial administration on hemostasis in animals with underlying disease processes are warranted.

Clopidogrel induced suppression of bovine platelet activation in vitro and a preliminary study of its effect on the development of Mannheimia haemolytica induced pneumonia.

We report here on the influence of the platelet antagonist clopidogrel (Plavix) on bovine platelet function. We first evaluated the capacity of clopidogrel to inhibit adenosine diphosphate (ADP)-stimulated platelet function in the bovine species, using an ex vivo approach with blood from treated animals. Platelets isolated from treated calves displayed rapid and consistent reduction in function (aggregation, thromboxane production) upon ADP, but not platelet activating factor (PAF), stimulation. We then examined the possibility that clopidogrel could influence Mannheimia haemolytica pneumonia pathobiology using an experimental challenge model. We were unable to detect significant differences between clopidogrel treated and untreated animals when challenged with intra-tracheal inoculation of M. haemolytica. There was a trend towards inhibition of platelet degranulation in the affected regions of lungs from clopidogrel treated calves, and pre-treated challenged animals had similar amounts of fibrin deposition and enhanced fibrous tissue formation in their lungs when compared with control counterparts.

Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development.

Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and antiangiogenic factors. The purpose of this study was to determine the expression of the antiangiogenic thrombospondin family and proangiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size (< 0.5 cm, small; 0.5-1.0 cm, medium; >1.0 cm, large). Thrombospondin (TSP) protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP colocalized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast with TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.

Comparative aspects of blood coagulation.

Blood coagulation is a basic physiological defense mechanism that occurs in all vertebrates to prevent blood loss following vascular injury. In all species the basic mechanism of clot formation is similar; when endothelium is damaged a complex sequence of enzymatic reactions occurs that is localized to the site of trauma and involves both activated cells and plasma proteins. The reaction sequence is initiated by the expression of tissue factor on the surface of activated cells and results in the generation of thrombin, the most important enzyme in blood clot formation. Thrombin converts soluble fibrinogen, via soluble fibrin monomers, into the insoluble fibrin that forms the matrix of a blood clot as well as exerting positive-feedback regulation that effectively promotes additional thrombin generation that facilitates the rapid development of a thrombus. Both spontaneous and trauma-induced haemorrhagic episodes can develop in all mammals with inherited or acquired abnormalities in one or more of the coagulant proteins. Experimental studies with plasma from a wide range of species have led to the conclusion that there are extensive differences in the rates of thrombin generation and fibrin formation among species. However, current evidence suggests that at least some of these quantitative differences are likely due to the use of non-species specific laboratory reagents. Although the individual proteins involved in the procoagulant pathways exhibit similar functions in all animals, differences in amino acid sequence cause incomplete homology and varying degrees of immunological cross-reactivity for the same protein across species.

A mathematical model of lipid-mediated thrombin generation.

Thrombin is an enzyme that is generated in both vascular and non-vascular systems. In blood coagulation, a fundamental process in all species, thrombin induces the formation of a fibrin clot. A dynamical model of thrombin generation in the presence of lipid surfaces is presented. This model also includes the self-regulating thrombin feedback reactions, the thrombomodulin-protein C-protein S inhibitory system, tissue factor pathway inhibitor (TFPI), and the inhibitor, antithrombin (AT). The dynamics of this complex system were found to be highly lipid dependent, as would be expected from experimental studies. Simulations of this model indicate that a threshold lipid level is required to generate physiologically relevant amounts of thrombin. The dependence of the onset, the peak levels, and the duration of thrombin generation on lipid was saturable. The lipid concentration affects the way in which the inhibitors modulate thrombin production. A novel feature of this model is the inclusion of the dynamical protein C pathway, initiated by thrombin feedback. This inhibitory system exerts its effects on the lipid surface, where its substrates are formed. The maximum impact of TFPI occurs at intermediate vesicle concentrations. Inhibition by AT is only indirectly affected by the lipid since AT irreversibly binds only to solution phase proteins. In a system with normal plasma concentrations of the proteins involved in thrombin formation, the combination of these three inhibitors is sufficient both to effectively stop thrombin generation prior to the exhaustion of its precursor, prothrombin, and to inhibit all thrombin formed. This model can be used to predict thrombin generation under extreme lipid conditions that are difficult to implement experimentally and to examine thrombin generation in non-vascular systems.

Expression and localization of thrombospondin-1 and -2 and their cell-surface receptor, CD36, during rat follicular development and formation of the corpus luteum.

Thrombospondin (TSP)-1 and -2 are extracellular matrix glycoproteins that are both antiangiogenic and important in regulating cellular development, differentiation, and function. To evaluate the expression of TSP in follicular and luteal development, ovarian cycles of Sprague-Dawley rats were synchronized and tissues collected daily at stages corresponding to the early antral, ovulatory, early luteal, and late luteal phases of the ovarian cycle. Immunohistochemistry and Western blot analyses demonstrated that TSP-1 protein and its receptor, CD36, were present in the early antral phase and were localized primarily to the granulosa cells of antral follicles. Both proteins were also present immediately after ovulation and were localized to the developing corpus luteum. Messenger RNA for TSP-1 showed a similar pattern, with expression at the early antral and ovulatory phases. Protein and mRNA expression for TSP-2 was relatively delayed compared to TSP-1, although TSP-2 also was expressed in granulosa cells. Both TSP-1 and -2 were increased in response to LH stimulation in vitro, whereas TSP-2 was suppressed by FSH. The temporal pattern of expression of TSP-1, -2, and CD36, which mirrors the active phases of angiogenesis in this experimental model, is compatible with a role for these proteins in the control of ovarian vascularization.

Thrombin generation and presence of thrombin receptor in ovarian follicles.

Prothrombin, once converted to its enzymatically active form (i.e., thrombin), induces a broad spectrum of cellular responses in both vascular and avascular tissues. Bovine ovarian granulosa cells isolated from healthy follicles of various sizes contain both prothrombin mRNA and immunologically reactive prothrombin that appears to be identical to prothrombin in follicular fluid and plasma. When tissue factor, the primary physiological activator of thrombin generation in plasma, is used to initiate thrombin formation, the profile of prothrombin-to-thrombin conversion is similar in follicular fluid and plasma. The conclusion that biologically functional prothrombin is synthesized by granulosa cells is further supported by evidence that mRNA for gamma-glutamyl carboxylase, an enzyme essential for the vitamin K-dependent posttranslational modification of prothrombin, is expressed in granulosa cells in a manner similar to prothrombin mRNA. Thrombin's biological effects are mediated through selective proteolytic cleavage and activation of specific receptors. Bovine granulosa cells possess thrombin receptor (PAR-1) mRNA, and as seen with prothrombin mRNA and gamma-glutamyl carboxylase mRNA, cells isolated from small follicles possess more PAR-1 mRNA than cells from large follicles. Thrombin receptor expression by cells in close proximity to an active thrombin-generating system suggests that these factors may be important mediators of cellular function in the ovarian follicle.

Determination of prothrombin in feline plasma.

Determination of the major common pathway protein prothrombin, a vitamin K-dependent protein synthesized in the liver, may be useful for identifying coagulopathies in cats with liver disease or vitamin K antagonism. In people with liver disease, prothrombin is more commonly and more severely decreased than other procoagulant proteins. The purpose of this study was to evaluate a commercial chromogenic assay(DiaPharma Group, West Chester, Ohio, USA) for the determination of prothrombin activity in plasma from healthy cats. The method involves the cleavage of prothrombin by Ecarin, a nonphysiologic enzyme activator that cleaves prothrombin to meizothrombin, which then interacts with a chromogenic substrate. Citrated (n = 20) and EDTA (n = 37) plasma samples from clinically healthy cats were tested using 100-fold and on occasion 200-fold dilutions. The assay was run according to manufacturer's specifications and the relative percentage prothrombin activity was calculated using standard curves generated from a feline citrated plasma pool and human reference plasma. Slope and regression values (r =.998) were similar for feline and human samples, suggesting that Ecarin cleaves prothrombin in both feline and human plasma in an analogous manner. The correlation between results obtained using feline vs human reference plasma was high for both citrated (r =.910) and EDTA samples (r =.998). When prothrombin was determined using human reference plasma, results from citrated feline plasma samples were 75.7% +/- 9.0% of normal compared to 91.6% +/- 7.0% of normal when the feline standard curve was used. Similar results were obtained using EDTA plasma. Our results indicate that the prothrombin chromogenic assay may be useful for evaluating one component of the hemostatic pathway in feline plasma. The prothrombin chromogenic assay utilizes routine instrumentation, requires small sample volume (5 microliter/assay), and may be used on EDTA plasma. To optimize sensitivity, the assay should be run using a standard curve generated with a feline plasma pool.