Steven M. Slack

Particle Diameter Influences Adhesion under Flow

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.

Chapter 2 Fluid dynamic and hemorheologic considerations

Abstract The previous edition of this work, published in 1985 (1), included a section on fluid dynamic and hemorheologic considerations in which the basic concepts of fluid flow, protein and cell transport, and blood rheology were presented. That material will not be reiterated in depth in this chapter but rather referred to as appropriate. Instead, the present chapter will attempt to consider the current status of research in this field and, to the extent possible, indicate those areas where our understanding is incomplete and in need of additional research efforts. Since the publication of the previous edition, our appreciation of the ability of fluid dynamic forces to influence blood-surface interactions has increased considerably. Progress has been made, not necessarily with respect to a detailed understanding of the manner in which fluid forces affect the behavior of materials in contact with blood in vivo, but rather in the growing recognition that the various blood and vascular cells exist in a dynamic environment in which flow contributes to an essential modulation of homeostatic events. In recognition of the complexity of the various biological processes that can occur in the presence of a graft implant or an organ replacement, a mechanistic rather than phenomenologic approach has slowly evolved that emphasizes the manner by which changes in fluid flow can affect the behavior of individual cells or proteins and their possible interaction with foreign materials. The approach here will be to consider the simplest case of blood flow and mass transport in a tube, as was done in the previous edition, and to delineate not only how basic characteristics of the system influence blood-material interactions, but also to distinguish where such simplifications can be misleading with respect to intravascular events or extracorporeal circuits. A description will be made of how the principles of blood flow and mass transport are being applied to such complicated events as cell proliferation and thromboembolism. In addition, the ability of shear stresses generated as a consequence of blood flow to modulate the biological activity of various cells will be discussed. Finally, several devices currently used to study the role of fluid flow in thrombotic and hemostatic processes will be described.

Platelets undergo phosphorylation of Syk at Y525/526 and Y352 in response to pathophysiological shear stress

Atherosclerotic plaques can lead to partial vascular occlusions that produce abnormally high levels of arterial wall shear stress. Such pathophysiological shear stress can promote shear-induced platelet aggregation (SIPA), which has been linked to acute myocardial infarction, unstable angina, and stroke. This study investigated the role of the tyrosine kinase Syk in shear-induced human platelet signaling. The extent of Syk tyrosine phosphorylation induced by pathophysiological levels of shear stress (100 dyn/cm(2)) was significantly greater than that resulting from physiological shear stress (10 dyn/cm(2)). With the use of phospho-Syk specific antibodies, these data are the first to show that key regulatory sites of Syk at tyrosines 525/526 (Y525/526) and tyrosine 352 (Y352) were phosphorylated in response to pathophysiological shear stress. Increased phosphorylation at both sites was attenuated by pharmacological inhibition of Syk using two different Syk inhibitors, piceatannol and 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (OXSI-2), and by inhibition of upstream Src-family kinases (SFKs). Shear-induced response at the Syk 525/526 site was ADP dependent but not contingent on glycoprotein (GP) IIb-IIIa ligation or the generation of thromboxane (Tx) A(2). Pretreatment with Syk inhibitors not only reduced SIPA and Syk phosphorylation in isolated platelets, but also diminished, up to 50%, the platelet-mediated thrombus formation when whole blood was perfused over type-III collagen. In summary, this study demonstrated that Syk is a key molecule in both SIPA and thrombus formation under flow. Pharmacological regulation of Syk may prove efficacious in treating occlusive vascular disease.

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°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 αIIbβ3) 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 αvβ3) 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 β3 subunit as GP IIb-IIIa, plays no role in SIPA. On the basis of these results, the assessment of platelet size distributions provides a sensitive and quantitative measurement for the study of SIPA.

Comparison of Shear Stress–Induced Platelet Microparticle Formation and Phosphatidylserine Expression in Presence of αIIbβ3 Antagonists

The use of platelet glycoprotein IIb-IIIa (&agr;IIb&bgr;3) antagonists is an accepted practice in the treatment of acute coronary syndromes. Recent studies have demonstrated that &agr;IIb&bgr;3 receptor antagonists are effective in inhibiting the procoagulant activity of platelets under static conditions. No investigation, however, has compared the ability of these platelet antagonists to inhibit platelet procoagulant activity, defined as an increase in phosphatidylserine (PS) expression, under conditions of shear stress. Thus, the goal of this study was to quantify the amount of microparticle formation and PS expression of platelets exposed to physiologic and pathophysiologic levels of shear stress in the absence and presence of three clinically approved parenteral &agr;IIb&bgr;3 antagonists (abciximab, eptifibatide, and tirofiban). Flow cytometric results demonstrated that although microparticle formation was significantly inhibited by all three antagonists, PS expression by sheared platelets was affected differently depending on the antagonist present. Specifically, abciximab suppressed PS expression compared with the saline control; both abciximab and eptifibatide significantly reduced PS expression compared with tirofiban; and tirofiban potentiated PS expression relative to the saline control at the highest shear stress. This is the first demonstration of differential regulation of platelet PS expression and, by inference, procoagulant activity in the presence of &agr;IIb&bgr;3 receptor antagonists under shear stress. The current results may have future importance in improving the design of platelet antagonists as well as defining the general role of fluid shear stress in platelet thrombus formation.

Hemocompatibility studies of surface-treated polyurethane-based chronic indwelling catheters

The objectives of this research were to evaluate and compare the interactions of several polyurethane-based central venous catheter materials with blood. Specifically, measurements of fibrinogen adsorption, platelet adhesion, kallikrein generation, and fibrinopeptide A (FPA) release were performed. The catheter materials examined in this study included: platinum-cured, 50 shore A durometer, barium sulfate-filled, silicone (SI); Tecoflex EG85A-B20 polyurethane (PU); PU catheters whose outer surface had been impregnated with ion beam-deposited silver atoms (AgI and AgII); PU catheters coated with a hydrophilic, polyacrylic acid polymer (UC); PU catheters coated with an air-cured PTFE emulsion (CS); and PU catheters coated with an aminofunctional dimethylsiloxane copolymer (JG). The time course of fibrinogen adsorption from plasma to the SI, JG, PU, and CS materials was similar, with CS exhibiting the least amount of adsorbed fibrinogen after 1 h (65 +/- 4.7 ng cm-2) and PU the greatest (144 +/- 16.5 ng cm-2). After 90 min of contact, AgI and AgII exhibited the greatest number of adherent platelets, levels that were approximately two to three times higher than those on the other catheter materials. With the exception of UC and PU, which caused kallikrein generation levels approximately half that of the positive (glass) control, little kallikrein formation was observed for any of the materials relative to the negative control. Finally, FPA generation was greatest using the SI, CS, and PU materials, with the latter causing the production of almost four times the amount of FPA as the negative control. This preliminary assessment of the hemocompatibility of the various catheters suggests that the surface treatments did not adversely affect their interactions with blood components; further investigations of these materials are therefore warranted in order to completely characterize their behavior prior to use in clinical situations.

A Computational Analysis of FXa Generation by TF:FVIIa on the Surface of Rat Vascular Smooth Muscle Cells

AbstractA 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.

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 bothKm andVmax. Values ofKm decreased from 151 to 16 nM as the shear rate increased from 25 to 2400 sec−1. Additionally, there was a twofold increase inVmax 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.

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials.

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.

Tissue factor expression by rat osteosarcoma cells adherent to tissue culture polystyrene and selected orthopedic biomaterials

Tissue factor (TF), a transmembrane glycoprotein expressed by numerous cell types, plays a critical role in the initiation of blood coagulation at sites of vascular injury. Activated products of the coagulation cascade may then enhance the inflammatory responses associated with wound healing. In the present investigation the ability of rat osteosarcoma (ROS) cells to express TF activity was examined following their growth on tissue-culture polystyrene (TCPS) and selected orthopedic biomaterials (titanium and zirconium alloys, and stainless steel). ROS cells exhibited significant TF activity as evidenced by the conversion of Factor X to Factor Xa in the presence of TF, Factor VIIa, and Ca2+. Factor Xa concentrations ranged from 1.0 fM per cell at 10 min to 6.0 fM per cell after 60 min. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited approximately twice the activity of non-stimulated cells when grown on TCPS but not on the metallic substrates. ROS cells (stimulated or unstimulated) adherent to the zirconium alloy generated lower amounts of Factor Xa compared to those bound to the other alloys and unstimulated cells grown on TCPS. These results indicate that ROS cells cultured on these synthetic surfaces differentially express procoagulant activity and that cells grown on TCPS, but not the metallic alloys, exhibit increased TF activity in response to stimulation by calcium ionophore. This procoagulant activity may potentiate subsequent inflammatory responses associated with the use of orthopedic biomaterials and thereby influence the tissue compatibility of the implant.