Patricia A. Gentry

Expression and Localization of Thrombospondin-1 and -2 and Their Cell-Surface Receptor, CD36, During Rat Follicular Development and Formation of the Corpus Luteum

Abstract 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.

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.

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.

Thrombin generation and presence of thrombin receptor in ovarian follicles.

Abstract 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 γ-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. Thrombins 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 γ-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.

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.

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).

Chapter 10 – Hemostasis

Publisher Summary This chapter reviews the comparative biochemistry of hemostasis. Hemostasis, the process of arresting the escape of blood from the vascular system, is integral to survival in animals. Hemostasis is regulated by a series of orchestrated events, and is dependent on the vessels through which blood flows, as well as numerous proteins and cells. Other systems in the body, such as the innate immune system and the inflammatory response, are also closely related to blood clotting and are influenced by the activation of hemostasis. The stepwise process that takes place to minimize blood loss and repair the injury includes initial vasospasm; platelet activation and plug formation; assembly and activation of the coagulation cascade factors; fibrin clot formation at the site of injury; and dissolution of the clot and vascular repair. This chapter begins with discussing the mechanisms of hemostasis and then elaborates laboratory assessment of hemostasis, as well as disorders of hemostasis. The chapter also describes how the dramatic variations in blood clotting in different species impact laboratory testing, response to therapeutic agents, downstream effects on other body systems, and the array of diseases that can occur.