ABSTRACT
BACKGROUND: P-selectin, expressed on platelets on activation, mediates rolling of platelets on endothelial cells, but its role in shear-induced platelet aggregation is not known. METHODS AND RESULTS: Platelets were exposed to either a single pulse (30 seconds) or 3 pulses (10 seconds) of high shear stress (150 to 200 dynes/cm(2)) each followed by low shear stress (10 dynes/cm(2)) for 4.5 minutes or 90 seconds, respectively, at 37 degrees C to resemble more closely in vivo conditions such as those in stenotic arteries. Under these conditions, platelet aggregation was significantly increased compared with low or high shear stress alone. Monoclonal anti-P-selectin antibodies inhibited shear-induced platelet aggregation, especially when induced by the combination of high and low shear stress, by approximately 70% and had an additive effect on the inhibition by abciximab (anti-glycoprotein (GP) IIb/IIIa antibody). However, anti-P-selectin antibody inhibited shear-induced platelet aggregation only at 37 degrees C, not at 22 degrees C, whereas abciximab inhibited shear-induced platelet aggregation at both 22 degrees C and 37 degrees C. This differential effect of anti-P-selectin antibody is explained by the finding that shear-induced P-selectin expression on platelets was observed mainly at 37 degrees C. CONCLUSIONS: These results indicate that pulsatile shear stress, which resembles flow conditions in stenotic arteries, induces significantly more platelet aggregation at 37 degrees C than monophasic shear stress. Under these conditions, we show a novel role for P-selectin in platelet aggregation distinct from that of GP IIb/IIIa, which may be of importance in the initiation of thrombosis associated with atherosclerotic lesions.
Subject(s)
P-Selectin/physiology , Platelet Aggregation/physiology , Analysis of Variance , Antibodies/immunology , Blood Platelets/metabolism , CD36 Antigens/immunology , Flow Cytometry , Humans , In Vitro Techniques , P-Selectin/immunology , Platelet Aggregation/immunology , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , TemperatureABSTRACT
Smooth muscle cells (SMC) are the major cellular component of the blood vessel wall and are continuously exposed to cyclic stretch due to pulsatile blood flow. This study examined the effects of a physiologically relevant level of cyclic stretch on rat aortic vascular SMC proliferation. Treatment of static SMC with serum, platelet-derived growth factor, or thrombin stimulated SMC proliferation, whereas exposure of SMC to cyclic stretch blocked the proliferative effect of these growth factors. The stretch-mediated inhibition in SMC growth was not due to cell detachment or increased cell death. Flow cytometry analysis revealed that cyclic stretch increased the fraction of SMC in the G(0)/G(1) phase of the cell cycle. Stretch-inhibited G(1)/S phase transition was associated with a decrease in retinoblastoma protein phosphorylation and with a selective increase in the cyclin-dependent kinase inhibitor p21, but not p27. These results demonstrate that cyclic stretch inhibits SMC growth by blocking cell cycle progression and suggest that physiological levels of cyclic stretch contribute to vascular homeostasis by inhibiting the proliferative pathway of SMC.
Subject(s)
Cell Cycle , Muscle, Smooth, Vascular/cytology , Animals , Aorta, Thoracic , Biomechanical Phenomena , Blood , Cell Death , Cell Division , Cells, Cultured , Cytokines/pharmacology , Flow Cytometry , Kinetics , Male , Muscle, Smooth, Vascular/physiology , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Rats , Rats, Sprague-Dawley , Thrombin/pharmacologyABSTRACT
Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles. In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec(-1) (shear stress approximately 375 dyne/cm2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation.
Subject(s)
Blood Platelets/drug effects , Peptide Fragments/pharmacology , Receptors, Thrombin/metabolism , Stress, Mechanical , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adenosine Diphosphate/pharmacology , Blood Coagulation/drug effects , Blood Platelets/physiology , Cell Membrane/drug effects , Cell Membrane/physiology , Cell Separation , Collagen/pharmacology , Crotalid Venoms , Drug Synergism , Epinephrine/pharmacology , Flow Cytometry , Humans , Platelet Activation/drug effects , Platelet Activation/physiologyABSTRACT
In order to further define the influence of microvessel diameter on intraluminal oxygen transport a previously described in vitro artificial capillary system was modified from a vessel diameter of 25 microns to 10 microns. Oxygen uptake and release rates were measured for hemoglobin solutions and red blood cell (Rbc) suspensions of the same overall hemoglobin concentration (10 g/dl). The modified apparatus was tested by comparing data for the hemoglobin solutions with predictive simulations from a validated mathematical model of oxygen transport. Preliminary data for oxygen uptake by Rbc suspensions flowing in 10 microns diameter capillaries are presented. As observed previously oxygen uptake is faster in hemoglobin solutions than in Rbc suspensions.
Subject(s)
Capillaries/metabolism , Oxygen/metabolism , Artificial Organs , Biological Transport , Erythrocytes/metabolism , Hemoglobins/metabolism , Humans , SolutionsABSTRACT
A mathematical model has been developed to predict oxygen transport by erythrocyte/acellular hemoglobin solution mixtures flowing in arteriolar-sized vessels (20 to 100 micron diameter). The model includes erythrocyte and extracellular hemoglobin solution phases, radial hematocrit and velocity gradients, axial convection, and radial diffusion of both oxygen and oxyhemoglobin. Model simulations were compared with experimental data from an in vitro capillary model where all of the geometric, physical, and transport parameters are known accurately. A new approach to shear augmentation of transport in 25-micron-diameter conduits was developed. Comparison of theory with experiment suggests that shear augmentation in this flow regime is primarily an extracellular phenomenon produced by cell-cell interactions. Negligible shear augmentation was seen in erythrocyte suspensions in plasma due to the relatively low solubility of oxygen in the plasma phase. Good agreement was found between the theoretical simulations and experimental data for release experiments even neglecting shear augmentation. However, treatment of shear augmentation significantly improved agreement between theoretical simulations and experimental data for oxygen uptake. The model was used to determine the effects on oxygen transport of varying extracellular hemoglobin concentration and extracellular hemoglobin oxygen binding characteristics. It is known that hemoglobin solutions transport oxygen more efficiently than erythrocyte suspensions of the same overall hemoglobin content. Model simulations show that erythrocyte/hemoglobin solution mixtures with 30% extracellular hemoglobin transport oxygen with virtually the same efficiency as pure hemoglobin solutions of the same overall hemoglobin content. Additional simulations predict that erythrocyte/hemoglobin solution mixtures transport oxygen more efficiently than Rbc suspensions, even if the extracellular hemoglobin has a high oxygen affinity.
Subject(s)
Erythrocytes/metabolism , Hemoglobins/metabolism , Microcirculation , Models, Biological , Models, Theoretical , Oxygen/blood , Animals , Biological Transport , Humans , Predictive Value of TestsABSTRACT
BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.
Subject(s)
Blood Platelets/cytology , CD18 Antigens/metabolism , Neutrophils/cytology , P-Selectin/metabolism , Abciximab , Adult , Antibodies, Monoclonal , Blood Platelets/chemistry , Blood Platelets/ultrastructure , Cations/metabolism , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Aggregation/drug effects , Cell Aggregation/physiology , Chelating Agents/pharmacology , Edetic Acid/pharmacology , Female , Flow Cytometry , Humans , Iloprost/pharmacology , Immunoglobulin Fab Fragments , Kinetics , Macrophage-1 Antigen/metabolism , Male , Microscopy, Electron , Middle Aged , Neutrophils/chemistry , Neutrophils/ultrastructure , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , Stress, Mechanical , VeinsABSTRACT
Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.
Subject(s)
Blood Platelets/metabolism , Purpura, Thrombotic Thrombocytopenic/metabolism , von Willebrand Factor/metabolism , Adenosine Diphosphate/pharmacology , Adult , Child , Child, Preschool , Female , Humans , Male , Middle Aged , P-Selectin/metabolism , Platelet Aggregation/drug effects , Platelet Count , Protein Binding , Recurrence , Ristocetin/pharmacologyABSTRACT
Oxygen transport behavior of erythrocyte/extracellular hemoglobin mixtures flowing in microvessels was studied as a model of hemoglobin-based oxygen carrier (HBOC) performance. An experimental in vitro 25-microm-diameter capillary model was used to provide detailed oxygen flux measurements for hemoglobin solutions, erythrocyte suspensions, and erythrocyte/hemoglobin solution mixtures. The experimental apparatus includes computerized data acquisition and control coupled to a dual wavelength microspectrophotomer. This apparatus had been previously validated by good agreement of experimental measurements with predictive mathematical models of oxygen transport for either erythrocyte suspensions or hemoglobin solutions. The experimental methodology was extended to measurement of oxygen transport in erythrocyte/hemoglobin solutions. The hemoglobin solutions consisted of either purified or gluteraldehyde polymerized bovine hemoglobin. Dose-response plots were generated by varying the extracellular to intracellular hemoglobin ratio while holding the overall hemoglobin concentration constant. Measurements were also made on unmixed erythrocyte suspensions and hemoglobin solutions to generate limiting cases for comparison. Direct comparison of experimental results showed that both types of hemoglobin solutions were substantially more efficient than erythrocyte suspension in uptake and release of oxygen. Increased extracellular hemoglobin concentration increased oxygen transport efficiency for both uptake and release, even when total hemoglobin concentration was held constant. When only 10% of the total hemoglobin was extracellular, approximately half of the increased efficiency of pure hemoglobin solutions was reached. When 50% of the total hemoglobin was extracellular, the increased efficiency was virtually equal to that of pure hemoglobin solutions.
Subject(s)
Blood Substitutes , Erythrocytes/metabolism , Hemoglobins/metabolism , Models, Biological , Oxygen/blood , Animals , Biological Transport, Active , Cattle , Hematocrit , Humans , In Vitro Techniques , SolutionsABSTRACT
Both nitric oxide (NO) and carbon monoxide (CO) are vessel wall-derived messenger molecules that cause platelet inhibition and vasodilation by activating guanylyl cyclase in target cells. Since vascular smooth muscle cells (SMCs) are exposed to shear and tensile stresses, this study examined the effects of these hemodynamic forces on the enzymes that generate NO and CO in SMCs. Monolayers of cultured rat aortic SMCs were subjected to shear stress using a modified cone and plate viscometer, or cyclic elongational stretch using a compliant silastic culture membrane. Shear stress stimulated time-dependent increases in mRNA and protein for inducible heme oxygenase-1 (HO-1), the enzyme which forms CO as a byproduct of heme degradation. The threshold level of shear necessary to induce HO-1 expression was between 5 and 10 dynes/cm2. In contrast, shear stress did not stimulate inducible NO synthase (iNOS) expression. Cyclic stretch also induced the expression of HO-1 but not of iNOS mRNA. Exposure of vascular SMCs to shear stress stimulated the production and release of CO as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. In addition, ADP-stimulated aggregation was inhibited in platelets exposed to sheared SMCs but not in platelets exposed to untreated control SMCs. Treatment of sheared SMCs with the HO-1 inhibitor, tin protoporphyrin-IX, blocked the antiaggregatory effect of the cells, whereas the iNOS inhibitor, methyl--arginine, had no effect. These results indicate that hemodynamic forces induce HO-1 gene expression and CO production in vascular SMCs, and that SMC-derived CO inhibits platelet aggregation. Thus, CO is a novel endogenous vessel wall-derived messenger molecule that may be selectively induced by hemodynamic forces to inhibit platelet reactivity and preserve blood fluidity at sites of vascular injury.
Subject(s)
Heme Oxygenase (Decyclizing)/biosynthesis , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/physiopathology , Animals , Cells, Cultured , Culture Media, Conditioned , Hemodynamics , Platelet Aggregation , Rats , Stress, MechanicalABSTRACT
Neutrophil emigration into inflamed tissue is mediated by beta 2-integrin and L-selectin adhesion receptors. Homotypic neutrophil aggregation is also dependent on these molecules, and it provides a model system in which to study adhesion dynamics. In the current study we formulated a mathematical model for cellular aggregation in a linear shear field based on Smoluchowski's two-body collision theory. Neutrophil suspensions activated with chemotactic stimulus and sheared in a cone-plate viscometer rapidly aggregate. Over a range of shear rates (400-800 s-1), approximately 90% of the single cells were recruited into aggregates ranging from doublets to groupings larger than sextuplets. The adhesion efficiency fit to these kinetics reached maximum levels of > 70%. Formed aggregates remained intact and resistant to shear up to 120 s, at which time they spontaneously dissociated back to singlets. The rate of cell disaggregation was linearly proportional to the applied shear rate, and it was approximately 60% lower for doublets as compared to larger aggregates. By accounting for the time-dependent changes in adhesion efficiency, disaggregation rate, and the effects of aggregate geometry, we succeeded in predicting the reversible kinetics of aggregation over a wide range of shear rates and cell concentrations. The combination of viscometry with flow cytometry and mathematical analysis as presented here represents a novel approach to differentiating between the effects of hydrodynamics and the intrinsic biological processes that control cell adhesion.
Subject(s)
Cell Adhesion , Cell Aggregation , Models, Biological , Neutrophils/physiology , Cell Size , Flow Cytometry , Humans , Integrins/metabolism , Kinetics , Mathematics , Microscopy , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/cytology , Neutrophils/drug effects , Receptors, Cell Surface/metabolism , Selectins/metabolism , Stress, Mechanical , ViscosityABSTRACT
The effects of intravenous Integrelin, an antagonist of platelet glycoprotein (GP) IIb-IIIa, were studied ex vivo in flow models of platelet adhesion/aggregation in patients undergoing angioplasty. Blood was collected from each patient before, during, and after a 24-h Integrelin infusion (0.75 micrograms/kg/min; plasma Integrelin levels, 312-759 ng/ml). The effects of Integrelin administered in vivo were evaluated by using two different models of platelet thrombus formation: (a) platelet adhesion onto von Willebrand factor (vWF)/collagen, followed by platelet aggregation, in a perfusion system; and (b) direct platelet aggregation induced by elevated levels of shear stress imposed by a cone-and-plate viscometer. Neither aspirin nor heparin, also given to the patients, affected platelet adhesion or aggregation in these flow models. In the perfusion studies, platelet adhesion to vWF/collagen I was not inhibited by in vivo Integrelin. In contrast, in each of the six patients studied by using blood collected after 45 min of Integrelin infusion, there was a decrease in the size of platelet aggregates compared with patient baseline samples. In the viscometer experiments, shear-induced platelet aggregation was reduced by 61-71% in samples collected 45 min into the Integrelin infusion (plasma Integrelin levels: 759 +/- 69 ng/ml) compared with baseline samples. Within 24-48 h after termination of the Integrelin, direct shear-induced platelet aggregation and platelet aggregation subsequent to adhesion returned to or near baseline in each of the patients studied. We conclude that Integrelin administered in vivo inhibits both platelet aggregation subsequent to initial platelet adhesion and direct shear-induced platelet aggregation under pathologic flowing conditions, After discontinuation of the drug, these inhibitory effects do not persist in vivo as long as the inhibitory effect on aggregation produced by c7E3 (the monoclonal antibody against GPIIb-IIIa).
Subject(s)
Blood Platelets/drug effects , Peptides/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Thrombosis/blood , Blood Platelets/physiology , Eptifibatide , Humans , Platelet AggregationABSTRACT
Shear stress-induced platelet aggregation requires von Willebrand factor (vWF), platelet glycoprotein (GP) Ib, GPIIb-IIIa, Ca2+, and adenosine diphosphate (ADP). Recent reports using vWF labeled with either 125I or fluorescein isothiocyanate (FITC) have demonstrated that in shear-fields, vWF binds to both GPIb and GPIIb-IIIa. The sequence of the vWF finding to the two platelet receptors has not been precisely determined in these reports. In this study, a flow cytometry technique using a primary anti-vWF antibody and a secondary FITC IgG antibody was used to measure shear stress-induced vWF binding to platelets. Washed normal platelets suspended at 50,000/microliters with purified large vWF multimers were exposed to laminar shear stresses of 15 to 120 dynes/cm2 for 30 sec. At this low platelet count, little or no aggregation occurred in the shear fields. A significant increase in post-shear vWF-positive platelets was consistently observed. Experiments with platelets from normal and severe von Willebrand's disease (vWD) (which lack plasma and platelet alpha-granule vWF) demonstrated that exogenous vWF predominately contributed to the platelet-vWF binding. Blockade of platelet GPIb with the monoclonal anti-GPIb antibody, 6D1, completely inhibited shear stress-induced platelet-vWF attachment. In contrast, blockade of GPIIb-IIIa with monoclonal anti-GPIIb-IIIa antibodies, 10E5, or c7E3, or with the GPIIb-IIIa-blocking tetrapeptide, RGDS had little or no inhibitory effect on platelet-vWF binding. These data demonstrate that the binding of vWF to GPIb is likely to be the initial shear-induced platelet-ligand binding event.
Subject(s)
Blood Platelets/metabolism , von Willebrand Factor/metabolism , Flow Cytometry , Hemorheology , Humans , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Stress, MechanicalABSTRACT
Homotypic adhesion o2 neutrophils stimulated with chemoattractant is analogous to capture on vascular endothelium in that both processes depend on L-selectin and beta 2-integrin adhesion receptors. Under hydrodynamic shear, cell adhesion requires that receptors bind sufficient ligand over the duration of intercellular contact to withstand hydrodynamic stresses. Using cone-plate viscometry to apply a uniform linear shear field to suspensions of neutrophils, we conducted a detailed examination of the effect of shear rate and shear stress on the kinetics of cell aggregation. A collisional analysis based on Smoluchowski's flocculation theory was employed to fit the kinetics of aggregation with an adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency. Adhesion efficiency increased with shear rate from approximately 20% at 100 s-1 to approximately 80% at 400 s-1. The increase in adhesion efficiency with shear was dependent on L-selectin, and peak efficiency was maintained over a relatively narrow range of shear rates (400-800 s-1) and shear stresses (4-7 dyn/cm2). When L-selectin was blocked with antibody, beta 2-integrin (CD11a, b) supported adhesion at low shear rates (< 400 s-1). The binding kinetics of selectin and integrin appear to be optimized to function within discrete ranges of shear rate and stress, providing an intrinsic mechanism for the transition from neutrophil tethering to stable adhesion.
Subject(s)
CD18 Antigens/blood , L-Selectin/blood , Neutrophils/physiology , Antibodies, Monoclonal/pharmacology , CD18 Antigens/biosynthesis , CD18 Antigens/immunology , Cell Adhesion/drug effects , Flow Cytometry , Humans , In Vitro Techniques , Kinetics , L-Selectin/biosynthesis , L-Selectin/immunology , Mathematics , Models, Biological , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Stress, Mechanical , ViscositySubject(s)
Blood Platelets/physiology , Hemorheology , Stress, Mechanical , Adult , Animals , Endothelium, Vascular/physiology , Hemorrhage/physiopathology , Hemostasis , Humans , Mice , Platelet Adhesiveness/physiology , Platelet Aggregation/physiology , Platelet Function Tests , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Signal Transduction , Thrombosis/physiopathology , von Willebrand Factor/physiologyABSTRACT
Elevated shear stress levels in pathologically stenosed vessels induce platelet activation and aggregation, and may play a role in the pathogenesis of arterial disease. Increased plasma catecholamine concentrations have also been implicated in the onset of acute coronary ischemic syndromes. This study was designed to examine the synergistic interaction of shear stress and epinephrine in the activation of platelets. Platelets (in PRP) sheared at 60 dyn/cm2 showed little or no aggregation unless pretreated with epinephrine. Pretreatment with 250 nM epinephrine followed by shear at 60 dyn/cm2 induced > 60% platelet aggregation. The specific alpha 2-adrenergic receptor antagonist yohimbine inhibited the synergistic aggregation, as did the ADP scavenging system phosphocreatine/creatine phosphokinase, indicating a three-way synergism with ADP. Chemical or monoclonal antibody blockade of von Willebrand factor (vWF) interactions with either platelet glycoprotein (Gp) Ib or Gp IIb/IIIa completely inhibited platelet aggregation induced by activating levels of shear stress alone. However, the combination of epinephrine and shear stress induced platelet aggregation that was blocked by 10E5, a monoclonal antibody that inhibits vWF binding to Gp IIb/IIIa, but not by aurin tricarboxylic acid or the monoclonal antibody 6D1, both of which inhibit vWF binding to Gp Ib. Synergistic platelet aggregation in response to epinephrine and shear stress was observed in washed platelets, platelet-rich plasma and whole blood in vitro, and also ex vivo following exercise to elevate endogenous levels of catecholamines. These results indicate that epinephrine synergizes with shear stress to induce platelet aggregation. This synergistic response requires functional Gp IIb/IIIa complexes, but is at least partially independent of vWF-Gp Ib interactions.
Subject(s)
Epinephrine/pharmacology , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIb-IX Complex/metabolism , von Willebrand Factor/metabolism , Calcium/blood , Cell Culture Techniques , Creatine Kinase/pharmacology , Exercise/physiology , Humans , Kinetics , Phosphocreatine/pharmacology , Stress, Mechanical , Yohimbine/pharmacologyABSTRACT
Intraluminal resistance to gas transport between the microcirculation and tissue was neglected for a half-century following the early work of Krogh. In recent years it has come to be understood that this neglect is seriously in error. This paper reviews the background for the long period of misdirection, and progress in placing the simulation of gas transport processes on a more accurate, quantitative basis.
Subject(s)
Blood-Air Barrier/physiology , Carbon Dioxide/metabolism , Microcirculation/physiology , Models, Cardiovascular , Oxygen/metabolism , Acid-Base Equilibrium/physiology , Anion Exchange Protein 1, Erythrocyte/metabolism , Biological Transport , Blood Flow Velocity , Hematocrit , Humans , Vascular Resistance/physiologyABSTRACT
Elevated levels of shear stress that occur in stenotic arteries may induce platelet aggregation and initiate thrombosis. Shear-induced platelet aggregation (SIPA) was studied in groups of ischemic stroke patients and normal subjects using a viscometric-flow cytometric technique. Twenty-three patients who sustained an ischemic stroke that was not of cardiac origin were included in this study, and were classified either as atherosclerotic (n = 15) or as lacunar (n = 8) stroke patients. The results show that shear stresses at the levels which occur in arteries partially occluded by atherosclerosis or vascular spasm strongly activate and aggregate platelets, and this response is much more pronounced in non-lacunar stroke patients who had documented atherosclerotic disease of their cerebral vessels. SIPA is not affected by the time of blood drawing after the onset of stroke suggesting that these platelet abnormalities are not transient but chronic. Furthermore, the extent of platelet activation detected by an anti-P-selectin monoclonal antibody and the proportion of neutrophil-platelet aggregates circulating in vivo are significantly higher in the atherosclerotic stroke patients studied at least one month after the onset of stroke. The results indicate that the enhanced platelet responses observed in atherosclerotic stroke patients are not consequences of ischemia, and therefore both platelet activation and elevated SIPA may be considered as important risk factors for stroke. The methodology developed in this work may be useful for characterization of platelet reactivity, and may contribute to our understanding of thrombotic mechanisms.
Subject(s)
Cerebrovascular Disorders/blood , Platelet Aggregation , Aged , Female , Flow Cytometry , Humans , Male , Middle Aged , Stress, MechanicalABSTRACT
In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were: 125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and 35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300-360 x 10(3)/microliters) of radiolabeled vWF was maximum at 60-120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.
Subject(s)
Blood Platelets/metabolism , Hemorheology , Platelet Aggregation/physiology , Platelet Glycoprotein GPIb-IX Complex/metabolism , von Willebrand Factor/metabolism , Antibodies, Monoclonal/pharmacology , Binding Sites , Blood Viscosity , Creatine Kinase/pharmacology , Humans , In Vitro Techniques , Iodine Radioisotopes , Phosphocreatine/pharmacology , Platelet Aggregation/drug effects , Radioligand Assay , Reference Values , Ristocetin/pharmacologyABSTRACT
BACKGROUND: Shear stress-induced platelet aggregation may initiate arterial thrombosis at sites of pathological blood flow. Shear stress-induced platelet aggregation is mediated by von Willebrand factor (vWf) binding to platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa. Tissue-type plasminogen activator (TPA) induces thrombolysis in coronary arteries through the local generation of plasmin. Plasmin also proteolyses GP Ib and plasma vWf. METHODS AND RESULTS: Because these effects could mitigate shear stress-induced platelet aggregation, we investigated the effect of fibrinolytic agents on platelet aggregation in response to a pathological shear stress of 120 dynes/cm2 generated by a cone-and-platen rotational viscometer. Plasmin inhibited shear stress-induced aggregation of washed platelets, and this was associated with a decrease in GP Ib. TPA, at concentrations > or = 2000 IU/mL, significantly inhibited shear stress-induced platelet aggregation of platelet-rich plasma without a decrease in platelet GP Ib. In plasma-platelet mixing experiments, we determined that the TPA effect was localized to plasma. Purified vWf multimer degradation by TPA (in the presence of exogenous plasminogen) was associated with the loss of the capacity of vWf to support shear stress-induced platelet aggregation. CONCLUSIONS: These results demonstrate that TPA inhibits platelet aggregation in response to pathological shear stress by altering the multimeric composition of vWf. This effect of TPA on shear stress-induced platelet aggregation may contribute, along with fibrinolysis, to the therapeutic effect of TPA in restoring blood flow during acute coronary artery thrombosis.
Subject(s)
Fibrinolysis , Platelet Aggregation , Fibrinolysin/pharmacology , Flow Cytometry , Humans , Platelet Membrane Glycoproteins/metabolism , Tissue Plasminogen Activator/pharmacology , von Willebrand Factor/metabolismABSTRACT
BACKGROUND: Elevated levels of shear stress such as those that occur in stenotic arterial vessels can directly activate and aggregate platelets and thus contribute to the pathogenesis of acute arterial thrombosis. This shear-induced platelet aggregation (SIPA) is mediated by von Willebrand factor binding to platelet membrane glycoprotein (GP) Ib and GPIIb/IIIa. The chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab) that binds selectively to GPIIb/IIIa is under clinical evaluation in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). This study was undertaken to investigate the effects on ex vivo SIPA of c7E3 Fab administered to patients undergoing PTCA. METHODS AND RESULTS: Six patients received aspirin (325 mg) and boluses of heparin (12,00o U) followed by c7E3 Fab 0.25 mg/kg. Blood collected from each patient before and after heparin treatment and at various time points after c7E3 Fab administration was subjected to laminar shear stress in a cone-and-plate viscometer. Flow cytometry was used to quantify the extents of platelet aggregation and of antibody binding to GPIIb/IIIa. Results indicate that c7E3 Fab injection resulted in a rapid, extensive blockade of GPIIb/IIIa receptors (98.6 +/- 0.2%) and a 50% inhibition of ex vivo platelet aggregation induced by shear stress. c7E3 Fab also completely abolished the formation of large platelet aggregates ("large" refers to particles > 10 microns in equivalent sphere diameter), which are presumably the aggregates of greatest clinical significance. Partial reversibility of the inhibition was noted within 2 days after drug administration, but even after 1 week, platelet function had not been fully restored. CONCLUSIONS: This study demonstrates that c7E3 Fab is a potent inhibitor of SIPA, which may be an important mechanism of its beneficial effect in the treatment of arterial occlusive diseases and in the prevention of thrombotic complications of coronary artery disease after angioplasty.