Platelet adhesion to a spinning surface.
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Animals↗
Biomedical subjects
V T Turitto
Publications and source records attributed to V T Turitto.
Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation.
The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm2 for 1 min at 37 degrees C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm2, whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin alpha v beta 3) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm2; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same beta 3 subunit as GP IIb-IIIa, plays no role in SIPA.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
A numerical analysis of factor X activation in the presence of tissue factor--factor VIIa complex in a flow reactor.
A mathematical model has been developed to investigate previously obtained experimental findings relating to the activation of factor X by surface-bound tissue factor--factor VIIa (TF:VIIa) in a tubular flow reactor. In those experiments, factor X was perfused through a microcapillary tube over a range of flow (shear) conditions and the activated product, factor Xa, was measured at the outlet of the tube using a chromogenic assay. In the present study, the steady-state convection-diffusion equation with Michaelis-Menten kinetics used to describe the reaction at the wall was numerically integrated using an implicit method based on linear systems of ordinary differential equations. The results from the numerical analysis indicated that shear rate directly affects both Km and Vmax. Values of Km decreased from 151 to 16 nM as the shear rate increased from 25 to 2400 sec-1. Additionally, there was a twofold increase in Vmax from 1.4 to 3.0 pmol/cm2/min as the shear rate increased from 25 to 300 sec-1. These findings are in contrast with classical enzyme behavior and imply a direct effect of fluid flow on the kinetics of factor X activation.
Factor VIIa↗
A computational analysis of FXa generation by TF:FVIIa on the surface of rat vascular smooth muscle cells.
A computational model was developed to investigate the contribution of classical mass transport and flow parameters to factor X (FX) activation by the tissue factor-factor VIIa complex (TF:VIIa) on one wall of a parallel-plate flow chamber. The computational results were compared to previously obtained experimental data for the generation of factor Xa (FXa) by TF:VIIa on the surface of cultured rat vascular smooth muscle cells. In this study, the complete steady-state convection-diffusion equation was solved using the commercial software package, FLUENT (Fluent Inc., Lebanon, New Hampshire). A user-defined subroutine interfaced with FLUENT implemented the surface reaction which was modeled using classical Michaelis-Menten reaction kinetics. The numerical solutions were obtained for 12 cases which used combinations of three wall shear rates and four reaction rates. The numerically obtained fluxes for a given reaction rate displayed a wall shear rate dependence which ranged from classical kinetic reaction control (no dependence) to pure diffusional control (maximum dependence). The experimental data, however, were not represented by numerical data generated using a single reaction rate. The three numerically obtained fluxes which corresponded most closely to the experimental fluxes were determined using three different Vmax values. This finding supports the hypothesis that there may be a direct effect of flow on the TF:VIIa complex or the cell membrane.
Algorithms↗
Platelet deposition at high shear rates is enhanced by high plasma cholesterol levels. In vivo study in the rabbit model.
We have studied the effects of high plasma cholesterol levels on platelet-vessel wall interactions under high shear rate conditions typical of the apex of stenotic arteries (2,600 sec-1). Hypercholesterolemia was induced by feeding rabbits a 0.5% cholesterol-rich diet for 60 days. Platelet deposition was studied by use of an annular perfusion chamber and de-endothelialized abdominal rabbit aortas as substrates. After ingestion of the atherogenic diet, the experimental group of animals developed severe hypercholesterolemia, platelets became more fluid as determined by steady-state fluorescence anisotropy (p less than 0.05), and red blood cell deformability was decreased (p less than 0.001) when compared with normal controls. The fatty acid composition of platelet membranes showed an increase in the percentage of the long-chain saturated fatty acids (palmitic, C16:0, and stearic, C18:0) that may account for the lower polyunsaturated/saturated fatty acid ratio observed in the hyperlipemic animals. Total platelet deposition was significantly increased (p less than 0.05) in the hyperlipemic group as compared with the control group at 5 minutes' perfusion time, becoming less evident at 20 minutes' perfusion time. Our results suggest that the presence of hyperlipidemia may contribute to acute thrombosis by enhancing platelet-vessel wall interaction.
Animals↗
Effect of aspirin and epinephrine on experimentally induced thrombogenesis in dogs. A parallelism between in vivo and ex vivo thrombosis models.
Thrombosis on the damaged or ruptured vascular wall in a stenotic coronary artery is believed to be the precipitating factor leading to unstable angina. Little is known about the nature of the interactions among platelets, fluid dynamic factors, and vessel wall properties under such conditions. In the present investigation we have compared two experimental models of thrombosis simultaneously in anesthetized dogs. The first was an in vivo model of unstable angina, in which a fixed circumflex coronary artery stenosis was produced and the resultant cyclic blood flow reductions (CFRs) through the vessel were investigated after infusion of aspirin and a combination of aspirin and epinephrine. As previously reported, aspirin inhibited the CFRs, but the continuous infusion of epinephrine reestablished the appearance of CFRs. The second was an ex vivo model, in which thrombus formation on a type III collagen surface was investigated in a parallel-plate perfusion system under controlled conditions of exposure time and flow; morphological evaluation of thrombus volume, platelet adhesion, and fibrin deposition was performed. The chamber was positioned in an extracorporeal shunt between the carotid artery and the jugular vein of anesthetized dogs and exposed to nonanticoagulated blood at a shear rate of 1,600 sec-1. Thirty minutes after establishment of the CFRs, a blood sample for platelet aggregation was collected and a bleeding time and a first ex vivo perfusion were performed. At the end of this perfusion, animals were subjected either to no treatment (n = 10) or to an intravenous bolus of 10 mg/kg aspirin (n = 7), and a second perfusion was conducted 30 minutes later. Additional untreated animals (n = 6) were given aspirin followed by a continuous intravenous infusion of 10 micrograms/ml epinephrine, and a third perfusion was conducted. Results with respect to platelet adhesion, thrombus volume, and fibrin deposition were similar in the two perfusions in untreated animals. Treatment with aspirin abolished the CFRs in all dogs and concomitantly reduced the ex vivo thrombus volume by 84% (p less than 0.01) without affecting platelet adhesion and fibrin deposition. Bleeding time increased by 40% (p less than 0.05), and collagen-induced platelet aggregation was virtually abolished (p less than 0.01). However, infusion of epinephrine in dogs after aspirin treatment restored the CFRs, and the ex vivo thrombus volumes were not statistically different from predrug values. Thus, the ex vivo model satisfactorily reflects the more complicated in vivo model events with respect to intracoronary thrombosis and substantiates the view that aspirin interrupts coronary thrombogenesis in the dog by interfering with platelet cohesion.
Animals↗
Functional behavior of vessels from pigs with von Willebrand disease. Values of platelet deposition are identical to those obtained on normal vessels.
Vessels from normal pigs and pigs with severe von Willebrand disease were exposed for up to 30 minutes to both nonanticoagulated and heparinized blood from normal pigs in an ex vivo perfusion system. Shear rates at the vessel surface were varied over a broad physiological range, gamma w = 212 to 3380 sec-1. The deposition of 111In-labeled platelets was determined by radiometric counting. For all shear rates and exposure times investigated, the levels of platelet deposition on de-endothelialized thoracic aorta of normal and von Willebrand disease pigs were not significantly different. Thus, the functional activity of the vessels correlated with the results obtained previously by immunofluorescence. Namely, the von Willebrand factor protein in the thoracic subendothelium of normal pigs is significantly diminished or absent and is comparable to the levels observed in von Willebrand disease pigs.
Animals↗
Role of von Willebrand factor in platelet interaction with an expanded PTFE surface.
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Animals↗