Ativação de plaquetas de eqüinos com laminite induzida e tratados com ketoprofeno, fenilbutazona e flunixin meglumina / Platelets activation in equine with induced laminitis and treated with ketoprophen, phenylbutazone and flunixin meglumin

Avaliou-se a ativação de plaquetas em 20 eqüinos com laminite induzida, tratados com ketoprofeno, fenilbutazona e flunixin meglumina. As alterações de plaquetas incluíram mudança de forma, alteração da relação entre os eixos maior e menor, aumento de perímetro, emissão de pseudópodes, aumento no número dealfa-grânulos e de grânulos de glicogênio e redução no número degama-grânulos. As plaquetas de eqüinos, quando ativadas, apresentaram perfil de organela diferente de plaquetas normais, e as drogas antiinflamatórias, não-esteroidais, demonstraram atividade na ativação plaquetária de eqüinos in vivo. O flunixin meglumina apresentou melhor atividade em modular a ativação plaquetária de eqüinos in vivo do que a fenilbutazona e o ketoprofeno.

c7E3 Fab inhibits low shear flow modulated platelet adhesion to endothelium and surface-absorbed fibrinogen by blocking platelet GP IIb/IIIa as well as endothelial vitronectin receptor--results from patients with acute myocardial infarction and healthy controls.

The c7E3 Fab reduces ischemic complications in patients undergoing high-risk coronary angioplasty or atherectomy. The present study investigated how c7E3 Fab inhibition of the platelet receptor glycoprotein IIb/IIIa and the endothelial vitronectin receptor affected platelet adhesion to endothelium and surface adsorbed fibrinogen under flow conditions. Platelet adhesion was examined using a stagnation point flow device with shear stress and shear rates up to 2.2 dynes/cm2 and 170 s(-1), respectively. Ex vivo adhesion was compared between two groups of patients with acute myocardial infarction (AMI) treated with angioplasty and stent implantation and a group of healthy controls. Only one AMI group received c7E3 Fab therapy. Patients in both groups were administered acetyl salicylic acid (ASA) and heparin. In AMI patients c7E3 Fab reduced platelet adhesion to adsorbed fibrinogen by 79% compared to AMI patients without c7E3 Fab treatment and by 74% compared to healthy controls. Thirty hours after termination of c7E3 Fab infusion adhesion had slightly recovered with an inhibition of 61% and 52% still present, respectively. Additionally, in vitro platelet adhesion to intact endothelium and to adsorbed fibrinogen was measured during superfusion with ADP stimulated platelet rich plasma of healthy controls to which c7E3 Fab was added at a final concentration fc of 20 microg/ml. In spite of ADP stimulation c7E3 Fab completely blocked platelet adhesion to adsorbed fibrinogen and, moreover, to intact endothelium. Preincubation of endothelial cells with c7E3 (fc = 20 [microg/ml) blocked adhesion of ADP-stimulated platelets by approximately 50%. Apart from the inhibition of platelet aggregation, c7E3 Fab added in vitro and given therapeutically in patients effectively blocks platelet adhesion to components of the injured as well as intact vessel wall under stagnation point flow conditions.

Impact of pancreas and kidney transplantation on determinants of blood and plasma viscosity.

Simultaneous pancreas and kidney transplantation (PKT) is associated with a deterioration of hemorheology. We investigated the determinants of plasma and blood viscosity (hct. 35%) after PKT (n = 49), in type 1 diabetes (n = 26) and in healthy controls (n = 24). Patients after PKT were subdivided due to their graft function (intact pancreas and kidney graft, n = 26; pancreas rejected, intact kidney graft, n = 23). We examined the correlations of total serum protein, albumin, fibrinogen, alpha 2-macroglobulin, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides with plasma and blood viscosity (hct. 35%) measured at a continuous shear range of 600-0.2 s-1 with a rotational viscometer (Haake, Germany). Total protein was strongly associated with plasma viscosity in all examined groups (r > 0.5, p < 0.03), it determined blood viscosity over the whole shear range in type 1 diabetic patients, but only at high shear rates after PKT (> or = 100 s-1). The strong association of albumin and blood viscosity in type 1 diabetes and in healthy controls (shear rates > or = 10 s-1) was not found after PKT. Fibrinogen correlated with plasma and blood viscosity (> or = 25 s-1) after PKT (p < 0.03) but no in type 1 diabetic patients or healthy controls. Alpha 2-macroglobulin correlated with plasma and high shear blood viscosity after PKT only after pancreas rejection, no correlation was found after successful PKT. It also correlated with plasma and blood viscosity at low and high shear rates in type 1 diabetes. Total cholesterol and low shear blood viscosity correlated positively in successfully transplanted patients (r > 0.44), but negatively after pancreas rejection (r > -0.44). No correlation was found in type 1 diabetic patients, a positive association was found in healthy controls for plasma and low shear blood viscosity. LDL cholesterol correlated negatively (after pancreas rejection) or positively (healthy controls) with low shear blood viscosity (p < 0.03) and positively with plasma viscosity. HDL cholesterol was negatively associated with high shear blood viscosity in all groups (p < 0.05), except after successful PKT, where no association was found. It did not correlate with plasma viscosity in any group. Triglycerides did not contribute significantly to blood viscosity in the examined groups. The metabolic alterations after PKT influence plasma proteins, lipids and corpuscular elements of blood with regard to their effect on rheology.

Adhesion of ADP-activated platelets to intact endothelium under stagnation point flow in vitro is mediated by the integrin alphaIIbeta3.

As we demonstrated earlier, platelets adhere to intact endothelium provided they are activated and convectively transported against the endothelial surface. To identify the platelet receptors involved we superfused cultured endothelium with activated platelet rich plasma (PRP) by means of the Stagnation Point Flow Adhesio- Aggregometer while blocking various platelet receptors. Inhibition was performed with the tetrapeptide RGDS, the non-peptide Ro-43-8857, or a monoclonal antibody directed against integrin alphaIIbeta3. Platelet deposition was video-recorded and quantified by image analysis. Infusion of RGDS or Ro-43-8857 into ADP-stimulated PRP completely prevented adhesion as well as subsequent aggregation. Interrupting the inhibitor infusion while ADP stimulation persisted, prompted adhesion and aggregation, demonstrating the reversibility of the inhibition. Platelet adhesion was irreversibly blocked by preincubation of the PRP with the moab against alphaIIbeta3. Its specific binding was confirmed by immunoelectron microscopy. Our results suggest that platelet adhesion to intact endothelium is mediated via platelet integrin alphaIIbeta3.

Spreading of platelets: a morphological marker for platelet reactivity in peripheral arterial occlusive disease.

Platelet-surface contact is the first step in thrombus formation. Platelet spreading makes this initial contact irreversible. On the other hand plasma lipids and fibrinogen have been described to activate platelets or promote adhesion. We therefore investigated whether platelet spreading under stagnation-point flow conditions correlated with plasma concentrations of cardiovascular risk factors such as fibrinogen and high density lipoprotein (HDL)-cholesterol. Platelet rich plasma (PRP) from patients with peripheral arterial occlusive disease and healthy controls was examined by means of the Stagnation-Point Flow Adhesio- Aggregometer (SPAA). The SPAA comprises a microscopic setup with a flow chamber that permits direct observation and quantitation of platelet deposition onto standardized surfaces. After the flow experiments the deposited platelets were analyzed morphometrically for the degree of spreading expressed as inverse circularity (1/C). 1/C was correlated over 2 X 2 tables of fibrinogen combined with plasma levels of HDL-cholesterol, each of which was divided into a low and high value group. The patient and control group differed significantly with regard to 1/C, i.e. patient platelets demonstrated more adhesive platelets with a more extensive degree of spreading. 1/C was inversely correlated with HDL-cholesterol and showed significant differences between the patient and the control group. Increased 1/C values were found when associated with high fibrinogen levels and simultaneously with low HDL-cholesterol concentrations. Platelet spreading shows a correlation with increased levels of independent plasmatic risk factors for thrombosis in PAOD patients. Obtained during stagnation-point flow, spreading seems to be a morphological marker for platelet hyperreactivity.

Influence of glycemic control on viscosity and density of plasma and whole blood in type-1 diabetic patients.

The hemorheological properties of blood play an important role in determining blood flow. Blood inertia, as characterized by blood density, controls blood flow in the large arteries, whereas blood viscosity becomes increasingly important with decreasing vessel diameter. In order to evaluate the impact of glycemic control on the rheological properties of blood, we examined viscosity (shear range: 600-0.2 s-1) and density of plasma and whole blood in 26 Type-1 diabetic patients and in 24 healthy controls, matched for age and sex. The diabetic subjects were subdivided into two groups according to their degree of glycemic control: 14 patients with good (HbA1c = 7.1 +/- 0.6%), and 12 patients with poor control (HbA1c = 8.7 +/- 0.7%). Diabetic patients as a whole did not differ from healthy controls in any of the rheological parameters. Subdivision of the patients due to their degree of glycemic control led to a marked rheological separation of Type-1 diabetic subjects with significantly lower plasma (P < 0.008) and whole blood viscosity (P < 0.03 at 10 and 25 s-1), and plasma density (P < 0.05) in well controlled patients. Compared with healthy controls, well controlled diabetic patients had significantly lower values of viscosity (P < 0.005) and density (P < 0.05) of plasma. Poorly controlled patients, on the other hand, did not differ from healthy controls in the examined rheological parameters. There seems to be a positive influence of good glycemic control on hemorheology in Type-1 diabetic patients.

Effect of fibrinogen substitution in afibrinogenemia on hemorheology and platelet function.

Fibrinogen substitution can correct bleeding in afibrinogenemia. We assessed the effect of fibrinogen substitution in a patient lacking immunoreactive fibrinogen. Fibrinogen and thrombin time were not measurable before, but became detectable within 30 min after substitution, parallelled by an increase in ADP-induced platelet aggregation from < 10% to 32%. Platelet adhesion, measured by Stagnation Point Flow Adhesio- Aggregometry, was not detectable prior to substitution but attained normal values thereafter. Scanning electron microscopy of adhering platelets revealed pseudopodia protrusion and spreading. Morphometry revealed two populations of spread platelets one of which demonstrated inhibited spreading as compared to healthy controls. Immunoelectron microscopy revealed normal GPIIb/IIIa receptor expression, both before and after substitution. Dynamic and kinematic viscosity of plasma and whole blood remained below the 99.9% confidence border of a healthy control group. In afibrinogenemia fibrinogen levels as low as 10% of normal concentration sufficed to normalize coagulation, platelet adhesion, and, partially, spreading.

Residence time in niches of stagnant flow determines fibrin clot formation in an arterial branching model--detailed flow analysis and experimental results.

Deposition of blood components in branching flow has been investigated primarily with regard to platelets. We instead examined thrombin-induced fibrin clot formation in separated laminar as well as turbulent branching flow. The most rapid clot growth and largest clot was obtained at the lowest inflow rate. Increased inflow reduced the clot size and turbulence completely prevented clot formation. Examination of corresponding flow conditions revealed the recirculation zone in laminar flow to be characterized by two stationary, counterrotating vortices. Niches of stagnant flow, exhibiting long residence times, low wall shear rates and characterized by convergent flow, were spared between the bulk flow and these vortices. Here, fibrin clot growth continued even when shear rates were increased more than 100-fold. Our results indicate that, in branching flow, the long residence times and convergent flow characteristic of flow niches rather than shear rate are critical for fibrin clot formation.

Flow mediated fibrin thrombus formation in an endothelium-lined model of arterial branching.

In vivo arterial thrombosis occurs preferentially at curvatures and branchings, i.e. regions of flow separation and recirculation where blood is retained orders of magnitude longer than within straight vessel sections. To examine the effect of such disturbed flow on endothelial thromboresistance glass T-branchings lined with endothelial cells from human umbilical cord veins (HUVEC) were perfused with buffered fibrinogen solution (3mg/ml). The flow was adjusted to form a large recirculation zone and flow conditions were determined beforehand by means of flow visualization via dye injection as well as by laser ultramicroscope anemometry. Thrombus formation, which was registered on-line by video and evaluated planimetrically, was induced by injection of thrombin at concentrations ranging from 0.3 to 2.0 units/ml. Fibrin thrombus growth always began within the flow niche at the point of flow separation and extended downstream along the wall and into the vessel lumen finally occluding up to 80% of the lumen. Light and electron microscopy revealed that overall thrombus form as well as the orientation of single fibrin fibers were correlated strictly to the prevailing streamlines. Despite the integrity of the endothelial lining fibrin thrombus formation occurred. The fibrin fibers closely contacted the endothelial surface. These results indicate that recirculation zones promote fibrin thrombus formation sufficient to obstruct the vessel lumen and that intact endothelium alone is insufficient in preventing adhesion of fibrin to its surface.

Estimation of shear stress-related blood damage in heart valve prostheses--in vitro comparison of 25 aortic valves.

The hemodynamics of heart valve prostheses can be reproducibly investigated in vitro within circulatory mock loops. By measuring the downstream velocity and shear stress fields the shear stresses which are clinically responsible for damage to platelets and red blood cells can be determined. The mechanisms of damage and the effects of shear stresses on blood corpuscles were investigated by Wurzinger et al. at the Aerodynamics Institute of the RWTH Aachen. In the present study, the above data are incorporated into a mathematical correlation, which serves as a basic model for the estimation of blood damage. This mathematical model was applied to in vitro investigations of 25 different aortic valve prostheses. The results were compared to clinical findings. In most cases agreement was good, indicating that this model may be directly applied to the clinical situation. This new method facilitates the estimation of clinically expected blood damage from in vitro measurements. It may be useful for the development and evaluation of new valve prostheses. By comparative evaluation of different valve types it also provides additional information to help the implanting surgeon select the optimum valve for his patient.

Histophysiology of the circulating platelet.

The role of platelets in hemostasis and thrombosis has been well established since Eberth's and Schimmelbusch's pioneering intravital microscopic experiments. A century ago the distinct features of the circulating "smooth disc" and the activated "spiny sphere" were described. Since then the underlying cell-biological processes transforming a harmless floating platelet into a sticky corpuscle, ready to release its stores of thrombogenic and atherogenic substances have been unveiled. However, its life-threatening capabilities have evolved from the necessity of preventing equally dangerous blood losses from a pressurized circulation system. As circulation depends on the liquid state of blood, the platelets and the molecules of the plasmatic coagulation system must circulate in an inactive state, to become activated at the site of "demand" to transform the liquid into a solid hemostatic plug. As in nucleated cells the plasma membrane, made up of a phospholipid bilayer with integrated glycoproteins, is the structure signalling environmental information to the platelet interior. Many of the receptors for stimulatory or inhibitory mediators elicit a cell-biological response via G-proteins and subsequent Ca2+ mobilization by IP3, or stimulation/inhibition of adenylate cyclase followed by changes in cytoplasmic levels of cyclic AMP. The supposed intracellular Ca2+ store of the platelets, the dense tubular system, also appears as the site of Ca2(+)-activated prostaglandin synthesis. Raised cytoplasmic Ca2+ levels promote the polymerization of G-actin to F-actin involved in the extension of pseudopodia in the course of "external shape change." Ca2(+)-activated myosin light-chain kinase phosphorylates myosin which becomes associated with F-actin, with the resulting acto-myosin complex providing the contractile force for "internal shape change," i.e., the centralization of organelles and for clot retraction later in hemostasis. More than by the three-dimensional actin cytoskeleton proper, the discoid shape typical of the nonstimulated platelet appears to be secured by a two-dimensional membrane skeleton of actin filaments anchored to membrane glycoproteins via actin-binding protein or spectrin and ankyrin. Although the microtubule coil has been confirmed as the main determinant of the mechanical stiffness of the platelet with biophysical techniques, its hitherto assumed role for the maintenance of the disc shape no longer appears tenable. The morphological phenomenon of the shape change comprises an alteration of membrane glycoproteins resulting in binding of "adhesive" molecules like fibrinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Fåhraeus-Vejlens effect: margination of platelets and leukocytes in blood flow through branches.

The distribution of platelets and leukocytes was studied in tube flow at T-junctions. The diameters were 200 microns for the main channel and 100 microns for the side branch, or 100 microns and 80 microns, respectively. The concentrations of platelets and leukocytes in the blood collected from the side branch and in the blood from the reservoir were measured using blood samples with different levels of fibrinogen. The concentration of platelets in the blood from the side branch was found to be about 1.2-1.5 times higher than in the reservoir. This ratio was found dependent on the feed hematocrit and fibrinogen concentration. The concentration of leukocytes in the blood from the side branch was about 0.9-1.4 times the value of the blood in the reservoir depending slightly on the fibrinogen concentration.

Effect of buflomedil on epinephrine-enhanced platelet aggregation in vitro and ex vivo.

The effect of buflomedil (4-(1-pyrrolidinyl)-1-(2,4,6-trimethoxyphenyl)-1-butanone) in vitro and ex vivo after intravenous and oral administration was tested upon epinephrine-enhanced platelet aggregation (PA) in platelet-rich plasma (PRP) prepared from heparinized blood of healthy volunteers. In vitro incubation of PRP with buflomedil in concentrations above 10 mumol/l resulted in a significant depression of PA to approximately one-third of the control. 30 min after a single intravenous dose of 2.5 mg/kg buflomedil a depression of epinephrine-enhanced PA to about 60% of the value before injection of the drug was observed. This effect wore off during a few hours and was no longer present 24 h thereafter. Oral ingestion of 600 mg/d buflomedil depressed PA to approximately two-thirds within 2 days, with a further decrease to some 50% after 6 days of intake. 2 days after termination of treatment epinephrine-enhanced PA had returned to premedication values. Unlike nonsteroidal antiinflammatory drugs buflomedil does not act through an inhibition of prostaglandin synthesis.

Plasma and platelet skimming at T-junctions.

Separation of plasma, erythrocytes, and platelets was studied in T-shaped bifurcations prepared by a casting technique. The diameters of the side and main channel were 100 microns and 200 microns, respectively. Small blood samples (about 20 microliters) were collected from the side channel for determination of erythrocyte and platelet concentrations which were compared to the corresponding values in the reservoir. Conversely to the well-known erythrocyte reduction due to plasma skimming a relative enrichment of platelets in the side branch was observed. The thickness of the plasma layer in the main channel was found to depend on the aggregation tendency of the erythrocytes for Reynolds numbers around 0.3.

Ultrastructural investigations on the question of mechanical activation of blood platelets.

The present study addresses the question whether platelets are activated by mechanical stresses as they occur in pathologically accelerated blood flow. Their potential mechanoreceptive properties were tested by subjecting human platelets to defined fluid mechanical forces for periods of milliseconds. Platelet activation was assessed by quantitative morphology, revealing besides activated platelets, irreversibly ballooned, lytic platelets. However, this morphometrically documented "shear activation" of platelets can be suppressed almost completely by the addition of enzyme-substrate systems, capable of removing adenosine diphosphate from the suspending medium. This is in keeping with a recent study from our laboratory showing that the behaviour of lactic dehydrogenase as marker for platelet lysis and beta-thromboglobulin as release marker refuted earlier data, suggesting a direct activation of platelets by shear. It is concluded that former evidence of "shear induced platelet activation" must be interpreted as the consequence of lytic damage to a small proportion of platelets.