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Biomedical subjects

L V McIntire

Publications and source records attributed to L V McIntire.

At least 109 records · Page 6Linked to original sources
PubMed1987

Rheological studies of erythrocyte-endothelial cell interactions in sickle cell disease.

The abnormal adherence of sickle erythrocytes to endothelial cells (EC) has been hypothesized to play a role in the initiation of vaso-occlusion in sickle cell anemia. We studied erythrocyte/endothelial cell interactions under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle cell trait (AS), mechanically injured normal, and "high reticulocyte control" red blood cells (RBC). Human umbilical vein endothelial cells grown to confluence on glass slides formed the base of a parallel plate flow chamber into which RBC suspensions were perfused at a constant flow rate, producing a wall shear stress of 1 dyne/cm2. Adhesion was monitored using video microscopy, and the number of adherent RBC was determined at ten-minute intervals during a wash out period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) RBC were also more adherent than control normal cells, but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less dense (younger) RBC were more adherent to EC than dense (older) cells for normal, SS and "high reticulocyte control" RBC. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of young RBC present. Increased adherence of young cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells. Microvascular occlusion is a major clinical problem in sickle cell anemia. This obstruction to blood flow could be due to decreased deformability of the cell and its inability to pass through small vessels. If this were the case it would be reasonable to expect that the most severely deformed sickle cells, the irreversibly sickled RBC (ISC), would play an important role in the initiation of vaso-occlusion. However, the number of circulating ISC is not well correlated with the frequency of painful crises and other microvascular occlusive phenomena. Recent evidence suggests that microvascular occlusion may be associated with increased adhesion of sickle cells to vascular endothelial cells. A strong correlation between erythrocyte adherence and clinical vaso-occlusive severity has been reported by Hebbel et al. Hoover et al. and Hebbel et al. demonstrated increased adhesion in static tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Anemia, Sickle Cell↗
PubMed1987

Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion.

The effect of flow on the adhesion of polymorphonuclear leukocytes (PMNL) to vascular endothelium was investigated using a parallel plate chamber with a well-defined flow field. Washed PMNL were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVEC) pretreated with formyl-methionyl-leucyl-phenylalanine (FMLP, 1 X 10(-7) mol/L) for five minutes. In other experiments HUVEC were pretreated with interleukin 1 (IL1,2 U/mL) for four hours. PMNL adhesion to stimulated and control HUVEC was measured over a physiologic range of wall shear stresses. PMNL adhesion to nylon-coated surface was also studied. At a wall shear stress of 0.98 dynes/cm2,283 +/- 37.3 PMNL/mm2 (mean +/- SEM) adhered to FMLP-treated HUVEC while 195 +/- 20.3 PMNL/mm2 adhered to control HUVEC. At 1.96 dynes/cm2, 68 +/- 14.1 PMNL/mm2 adhered to FMLP-treated HUVEC and 42 +/- 6.0 PMNL/mm2 adhered to control HUVEC. At 3.92 dynes/cm2, virtually no PMNL adherence was noted on either control or FMLP-treated HUVEC. On IL 1-treated HUVEC at 1.96 dynes/cm2, 371 +/- 25.8 PMNL/mm2 adhered while 28 +/- 2.9 PMNL/mm2 adhered to control HUVEC. PMNL adhesion to IL 1-treated and control HUVEC dropped to 10.2 +/- 3.8 and 6.8 +/- 3.5 PMNL/mm2, respectively, at 3.01 dynes/cm2. The effect of flow on PMNL adhesion appears to be an important factor in determining the outcome of the PMNL/HUVEC adhesive interaction under these experimental conditions.

Cell Adhesion↗
PubMed1986

Platelet microtubules in clot structure formation and contractile force generation: investigation of a controversy.

Whether platelet microtubules are involved in clot retraction/contraction has been controversial. To address this question we have simultaneously measured two clotting parameters, clot structural rigidity and isometric contractile force, using a rheological technique. For recalcified PRP clots these two parameters began rising together at about 15 min after CaCl2 addition. In the concentration range affecting microtubule organization in platelets, colchicine, vinca alkaloids and taxol demonstrated insignificant effects on both clotting parameters of a recalcified PRP clot. For PRP clots induced by adding small amounts of exogenous thrombin, the kinetic curves of clot rigidity were biphasic and without a lag time. The first phase corresponded to a platelet-independent network forming process, while the second phase corresponded to a platelet-dependent process. These PRP clots began generating contractile force at the onset of the second phase. For both rigidity and force parameters, only the second phase of clotting kinetics was retarded by microtubule affecting reagents. When PRP samples were clotted by adding a mixture of CaCl2 and thrombin, the second phase clotting was accelerated and became superimposed on the first phase. The inhibitory effects of microtubule affecting reagents became less pronounced. Thrombin clotting of a two-component system (washed platelets/purified fibrinogen) was also biphasic, with the second phase being microtubule-dependent. In conclusion, platelet microtubules are important in PRP clotted with low concentrations of thrombin, during which fibrin network formation precedes platelet-fibrin interactions. On the other hand they are unimportant if a PRP clot is induced by recalcification, during which the fibrin network is constructed in the presence of platelet-fibrin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1986

Mechanical effects on endothelial cell morphology: in vitro assessment.

Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function.

Animals↗
PubMed1986

Visualization and analysis of mural thrombogenesis on collagen, polyurethane and nylon.

Visualization and analysis of mural thrombogenesis on collagen-coated glass, polyurethane and nylon was discussed. Epi-fluorescent video microscopy was used to visualize thrombotic events at a protein or polymer surface in contact with flowing whole blood. Digital image processing was used to analyse the real-time microscopic images obtained, resulting in measurements of morphological features and the number of platelets that compose each thrombus. The three-dimensional structures of the thrombi were also estimated. A photodiode was used to measure integrated end-point platelet accumulation at different axial positions along the surface. Additionally, the convection-diffusion equations were solved to estimate the concentrations of adenosine diphosphate, thromboxane A2, and thrombin generated by activated platelets at the blood-contacting surface.

Adenosine Diphosphate↗
PubMed1986

Platelet active concentration profiles near growing thrombi. A mathematical consideration.

When blood contacts foreign material surfaces, platelets usually adhere and form aggregates on those surfaces, generating mural thrombi. The mechanism of mural thrombogenesis is not completely understood, but one hypothesis states that the local release of certain platelet-active substances from the platelets composing an initial small thrombus stimulates additional platelet recruitment to that thrombus, resulting in growth of the cell aggregate. The purpose of this paper is to investigate the feasibility of this hypothesis. Concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), and thrombin were computed in the vicinity of growing model thrombi 10 and 20 micron long. Wall shear rates of 100, 500, and 1,500 s-1 were considered for blood flowing through a thin rectangular slit 200 micron wide coated with collagen, a predominant subendothelial protein. The local concentrations of ADP and TxA2 were marginally large enough to stimulate platelet activation individually, while local thrombin levels can be much greater than required for stimulation. Antithrombin III, a natural thrombin inhibitor, did not significantly reduce the thrombin concentrations, but antithrombin III accelerated by heparin greatly reduced the local thrombin concentrations. The reduced thrombin levels may, however, still be large enough to activate platelets.

Adenosine Diphosphate↗
PubMed1986

Platelet modulation of polymorphonuclear leukocyte shear induced aggregation.

A cone and plate viscometer and Coulter Counter were used to study platelet modulation of polymorphonuclear leukocyte (PMNL) aggregation caused by controlled shear stress. As an index of aggregation, the large-particle percentage (LPP) was calculated. This represents the ratio of aggregated cell count to total cell count. PMNL suspensions in buffer (1.0 X 10(7) cells per milliliter, final concentration) did not show any aggregate formation at shear stresses below 150 dynes/cm2 for one minute exposure time (LPP less than 3%). However, there was PMNL aggregation in mixed PMNL and platelet-rich plasma suspensions in this shear stress range. Supernatant plasma from sheared platelets initiated PMNL aggregation at moderate shear stress (150 dynes/cm2 for one minute; LPP, 20.3% +/- 2.5%). In contrast, platelet release factors, such as adenosine diphosphate (2 mumol/L) and serotonin (2 mumol/L) did not cause PMNL aggregation (LPP, 2.9% +/- 1.2% and 3.3% +/- 0.8%, respectively). The use of a cyclo-oxygenase inhibitor (acetylsalicylic acid, 50 mumol/L) did not suppress the aggregation of PMNLs after shear (LPP, 20.1% +/- 2.4%). However, preincubation with nordihydroguaiaretic acid (10 mumol/L), an inhibitor of C-5 and C-12 lipoxygenase, and 6,9-deepoxy-6,9-(phenylimino)-6,8-prostaglandin I1 (U-60257, 10 mumol/L), an inhibitor of C-5 lipoxygenase in human leukocytes, suppressed this aggregation (LPP, 9.1% +/- 2.5% and 10.4% +/- 3.2%, respectively). Also, the formation of lipoxygenase products (5-HETE, 12-HETE, 15-HETE, and LTB4) activated by shear stress was documented by reversed phase-high-performance liquid chromatography (RP-HPLC). These data support the possibility of a cooperation between platelets and leukocytes in shear-induced PMNL aggregation that is dependent on C-12 or C-5 lipoxygenase activity, or both.

Arachidonic Acid↗
PubMed1985

Flow effects on prostacyclin production by cultured human endothelial cells.

Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow. The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.

Blood Circulation↗
PubMed1985

Role of the membrane concanavalin A binding site in platelet-fibrin interactions.

Concanavalin A was employed to study the role of platelet membrane glycoproteins in platelet-fibrin interactions during clot formation. A rheological technique was used to study the interactions, measuring the clot rigidity and platelet contractile force simultaneously during the formation of network structure. Concanavalin A lowered the clot rigidity and contractile force of a platelet-rich plasma clot by a small extent. Plasma glycoproteins probably compete with platelet membranes for concanavalin A binding in platelet-rich plasma. Both native concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) lowered the clot rigidity and contractile force of a washed platelet-fibrin clot dramatically, almost down to those values found for fibrin clots. Inhibition studies with alpha-methyl-D-mannoside indicated that the concanavalin A effects were specific for the concanavalin A binding capacity to platelets. The effects of native concanavalin A on platelet-fibrin clots were only partially reversible, while the succinyl concanavalin A effects were completely reversible. The observed concanavalin A effects are probably mainly due to concanavalin A binding to platelet membrane glycoproteins. The concanavalin A binding site appears to play an important role in the fibrin binding to platelets.

Binding Sites↗
PubMed1984

The gelation kinetics of platelet extracts.

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca2+ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca2+ concentration up to about 2 microM. At 4-15 microM free Ca2+, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca2+ concentrations. Trifluoperazine (50 microM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca2+ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K+, ATP, cytochalasin E and colchicine. K+ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelation, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 microM) and colchicine (1 mM) interfered with the gelation process.

Adenosine Triphosphate↗
PubMed1984

Response of cultured endothelial cells to steady flow.

A system has been developed for subjecting endothelial cell monolayers to prolonged steady flow while maintaining normal culture conditions. Cloned bovine endothelial cells were grown to confluence on one wall of a square glass tube which was then incorporated in the flow circuit. Flow rates of 19-21 ml/min were sustained for periods of 6-45 hr, subjecting the cells along the center line of the wall of the tube to a maximum shear stress of 34 dyn/cm2. The cells in all the experiments remained attached and viable when subjected to this shear stress. Photographic data from experimental runs were qualitatively assessed for changes in cell morphology, confluence, and orientation and were compared to data from matched stationary controls. Five experiments were chosen for quantitative morphometric analysis. In three experiments, the cells showed elongation with their long axes aligned with the direction of flow in 6.5, 21, and 22 hr. In the other experiments, either the cells formed a swirling pattern or no change in morphology was apparent. Although cell shape (form) changed in response to shear stress, cell area remained unaffected by exposure to flow.

Animals↗
PubMed1984

Mechanical and biochemical aspects of leukocyte interactions with model vessel walls.

Leukocytes in flowing blood are continually undergoing collisions with the blood vessel walls. Whether these collisions result in adherence depends on a delicate balance between the fluid mechanical drag force, which tends to dislodge the PMNL, and the adhesive force generated at the area of contact with the endothelium. Local blood flow rate controls the first of these forces, with the important parameter being the velocity gradient at the wall (wall shear rate). The detailed morphology of the endothelial cell and the PMNL upon collision and the biochemical state of these cells determine the adhesive force, because this force is a product of the strength of interaction times the area of contact. If a leukocyte flattens out or spreads on the vessel wall surface, it will reduce the hydrodynamic drag and increase the area of contact, leading to a more stable adhesion. In the normal circulation, a significant fraction of the PMNL appear to be attached to the endothelium, particularly in the low flow venules. This leukocyte fraction is what hematology texts refer to as the marginal pool. The adherent PMNL are a dynamic population in the sense that some return to the circulation, some move through the endothelial cell mono-layer into the extravascular space, and others remain "attached" to the endothelial cells but roll along the surface in the flow direction. An equilibrium number is maintained under normal conditions by recruitment from the circulating pool. Increased blood flow rate will increase the hydrodynamic force, tending to dislodge the PMNL, but will also (at least in large vessels) increase the number of collisions of circulating PMNL with the vessel wall by increasing the effective "diffusion" coefficient of the leukocyte. Thus if one studied the kinetics of PMNL adhesion on an initially clean endothelial monolayer surface under flow, increasing the whole blood flow rate would probably increase in initial rate of attachment but may decrease the equilibrium number of adherent leukocytes (Fig. 5). The degree of cooperation in vivo between PMNL and endothelial cells required to enable the leukocytes to perform their bactericidal functions is fascinating. This type of interdependency also appears to be necessary in the inflammatory response [Wedmore and Williams, 1981]. Fluid mechanical forces in addition to biochemical events are crucial in regulating these interaction processes in vivo. In vitro models for examining PMNL adhesion should include control of the local fluid mechanics.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Flow Velocity↗
PubMed1984

Characteristics of shear-induced aggregation in whole blood.

A cone-and-plate viscometer was used to study shear-induced aggregation in whole blood. Aggregate size distribution curves were measured from samples subjected to controlled shear rates for varying time intervals. Blood samples in heparin, but not in citrate, developed aggregates at shear rates below 500 sec-1. These aggregates disintegrated at 1000 to 2000 sec-1. There appears to be a donor-specific threshold shear rate, above which significant amounts of shear-induced aggregation were found. For normal donors this threshold is in the range of 2000 to 3000 sec-1 for exposure times of 1 min. The extent of aggregation and the stability of aggregates formed increased with both shear rate and the shear time. Above the threshold shear rate, less than 6 sec exposure time was sufficient to trigger aggregation. Aggregates generated by exposing blood to shear above these levels for 30 to 60 sec were stable for at least 5 min. Blood in heparin was more sensitive to shear than that in citrate. Whole blood was more sensitive to shear than platelet-rich plasma. An ADP-utilizing enzyme system, creatine phosphate/creatine phosphokinase, was effective in reducing both sensitivity to and stability of shear-induced aggregation. Creatine phosphate alone was partially effective, whereas creatine phosphokinase alone was ineffective. This indicates that ADP released from cellular components in blood plays a positive role in shear-induced aggregation.

Creatine Kinase↗
PubMed1983

Importance of plasma fibronectin in determining PFP and PRP clot mechanical properties.

Plasma fibronectin has been shown to be incorporated into fibrin clots. The serum concentration of fibronectin is 20-50% lower than the plasma concentration (1). Grinnell and Feld, using an indirect immunofluorescence analysis on blood clots prepared on plastic strata, demonstrated that the fibrin-platelet meshwork was covered with a uniform coating of fibronectin (2). Fibronectin binds to fibrin by either non-covalent attachment or covalent crosslinking. The covalent crosslinking of fibronectin to the fibrin alpha-chain is via an epsilon-(gamma-glutamyl)lysine linkage, mediated by Factor XIIIa (3). Each fibronectin molecule has two such binding sites. Thus it appears that fibronectin may play a role in blood coagulation. It has been suggested that the mechanical properties of fibrin clots may be enhanced by the presence of fibronectin crosslinking (2). Kamykowski, et al. (4) have shown that the shear modulus of a ligated clot formed from purified fibrinogen can be either increased or decreased if fibronectin is present, depending on the ionic strength and the pH during network formation. Although fibronectin is associated with the network structure in plasma clots, the mechanical role of this fibronectin has not been established. In this study we examine quantitatively the effect of plasma fibronectin on the dynamic rigidity moduli of fibrin clots formed from platelet free plasma (PFP), as well as from platelet rich plasma (PRP).

Biophysical Phenomena↗
PubMed1983

Kinetics of platelet adhesion and thrombus growth.

Epifluorescent microscopy was used to monitor the adhesion of platelets and the growth of platelet aggregates on collagen-coated glass tubes perfused with whole blood. The maximum basal length and width of the aggregate size increased linearly with time, growing symmetrically transverse to the direction of flow and asymmetrically in the plane longitudinal to the direction of flow. Aggregates had elliptical bases, with the major axis parallel to the direction of blood flow. These studies provide an experimental approach to studies of the kinetics of platelet interaction with artificial surfaces and give further support to the concept that blood flow has a major effect on the development of platelet thrombi.

Collagen↗
PubMed1982

Effects of colchicine and vinblastine on platelet contractility and release.

Pre-incubation of normal platelet-rich plasma for 30 minutes at 37 degrees C with concentrations of colchicine (2.5 x 10(-4)M) or vinblastine (1.1 x 10(-4)M) which disrupt platelet microtubles did not inhibit aggregation induced by ADP or collagen. Even following pre-incubation of platelets with a 10-fold higher concentration of colchicine (2.5 x 10(-3)M), platelets released 14C-serotonin and aggregated in response to collagen. In the presence of a 10-fold higher vinblastine concentration (1.1 x 10(-3)M), platelets lysed and released 51Cr. Dynamic viscoelastic measurements on recalcified platelet-rich plasma in a Weissenberg rheogoniometer demonstrated that neither colchicine (2.5 x 10(-4)M to 5 x 10(-3)M) nor vinblastine (1.1 x 10(-4)M) interferes with the initiation, rate of generation, or extent of contractile force produced in platelet-fibrin clots. These results indicate that polymerized tubulin is not essential for collagen-induced release of platelet dense granule contents. In addition, intact tubulin polymers are not required to support the platelet surface configuration necessary for platelet-fibrin adherence and the platelet contractile events which result in platelet-fibrin clot retraction.

Blood Platelets↗