Investigation of platelet margination phenomena at elevated shear stress

Thrombosis is a common complication following the surgical implantation of blood contacting artificial organs. Platelet transport, which is an important process of thrombosis and strongly modulated by flow dynamics, has not been investigated under the shear stress level associated with these devices, which may range from tens to several hundred Pascal.The current research investigated platelet transport within blood under supra-physiological shear stress conditions through a micro flow visualization approach. Images of platelet-sized fluorescent particles in the blood flow were recorded within microchannels (2 cm x 100 microm x 100 microm). The results successfully demonstrated the occurrence of platelet-sized particle margination under shear stresses up to 193 Pa, revealing a platelet near-wall excess up to 8.7 near the wall (within 15 microm) at the highest shear stress. The concentration of red blood cells was found to influence the stream-wise development of platelet margination which was clearly observed in the 20% Ht sample but not the 40% Ht sample. Shear stress had a less dramatic effect on the margination phenomenon than did hematocrit. The results imply that cell-cell collision is an important factor for platelet transport under supra-physiologic shear stress conditions. It is anticipated that these results will contribute to the future design and optimization of artificial organs.     

Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow

The kinetics of adhesion of platelets to fibrinogen (Fg) immobilized on polystyrene latex beads (Fg-beads) was determined in suspensions undergoing Couette flow at well-defined homogeneous shear rates. The efficiency of platelet adhesion to Fg-beads was compared for ADP-activated versus "resting" platelets. The effects of the shear rate (100-2000 s(-1)), Fg density on the beads (24-2882 Fg/microm(2)), the concentration of ADP used to activate the platelets, and the presence of soluble fibrinogen were assessed. "Resting" platelets did not specifically adhere to Fg-beads at levels detectable with our methodology. The apparent efficiency of platelet adhesion to Fg-beads readily correlated with the proportion of platelets "quantally" activated by doses of ADP, i.e., only ADP-activated platelets appeared to adhere to Fg-beads, with a maximal adhesion efficiency of 6-10% at shear rates of 100-300 s(-1), decreasing with increasing shear rates up to 2000 s(-1). The adhesion efficiency was found to decrease by only threefold when decreasing the density of Fg at the surface of the beads by 100-fold, with only moderate decreases in the presence of physiologic concentrations of soluble Fg. These adhesive interactions were also compared using activated GPIIbIIIa-coated beads. Our studies provide novel model particles for studying platelet adhesion relevant to hemostasis and thrombosis, and show how the state of activation of the platelet and the local flow conditions regulate Fg-dependent adhesion.     

Platelet Dynamics in Three-Dimensional Simulation of Whole Blood

A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics.     

Platelet Dynamics in Three-Dimensional Simulation of Whole Blood

A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics.     

Discrimination between red blood cell and platelet components of blood clots by MR microscopy

Magnetic resonance imaging (MRI) of pulmonary emboli obtained ex vivo, verified by immunohistochemistry, showed that platelet layers display brighter signal intensity than areas containing predominantly red blood cells (RBC) in T (1)-weighted MRI. These results were surprising since platelets do not contain paramagnetic haemoglobin that would enhance magnetic relaxation. Our assumption was that the fibrin meshwork areas with entrapped RBC retain abundant extracellular space filled with serum, whereas platelets regroup into tight aggregates lacking serum, essentially mimicking solid tissue structure, rich with cellular proteins that enhance T (1)-relaxation. Our hypothesis was examined by MRI and NMR relaxometry of in vitro RBC suspensions and sedimented platelets, as well as by MRI of model clots and pulmonary emboli obtained ex vivo. Pure sedimented platelets exhibited shorter proton spin lattice relaxation times (T (1) = 874 +/- 310 ms) than those of venous blood of a healthy male with 40% haematocrit (T (1) = 1277 +/- 66 ms). T (1)-values of RBC samples containing high haematocrit (>/=80%) resembled T (1) of platelet samples. In T (1)-weighted spin-echo MRI echo time and repetition time (TE/TR = 10/120 ms) the ratio of signal intensities between a non-retracted whole blood clot (with a haematocrit of 35%) and a pure platelet clot was 3.0, and the ratio between a retracted whole blood clot with an estimated haematocrit of about 58% and a pure platelet clot was 2.6. We conclude that T (1)-weighted MRI can discriminate between platelet layers of thrombi and RBC-rich areas of thrombi that are not compacted to a haematocrit level of >/=80%.     

Platelet near-wall excess in porcine whole blood in artery-sized tubes under steady and pulsatile flow conditions

Platelet margination (enhanced platelet concentration in the near wall region of a blood vessel) has been well documented in small vessels. In artery-sized vessels margination has only been demonstrated in one study, using ghost cell suspensions and under relatively non-physiologic conditions of steady flow and 50 cm development length. Local sampling experiments were performed to confirm platelet margination in artery-sized stainless steel tubes, for a typical anatomical length and under pulsatile flow, using fresh EDTA-anticoagulated porcine whole blood (N=21). Experiments were designed using three-dimensional Computational Fluid Dynamics (CFD) to model the sample region with greater fidelity. Steady flow experiments in 50 cm long tubes verify published laser Doppler measurements of platelet margination in 3 mm ID tubes at normal arterial shear rate (500 s(-1). Margination persists under pulsatile flow conditions (63.8 pulses/min), but in steady flow at length of 10 cm, margination is reduced. Platelet margination ratio (the ratio of the platelet concentration near the wall to bulk average platelet count) ranges from 1.21 to 2.48. No significant effects of calculated sampling thickness (20 microm and 50 microm) or pulsatility were detected. Hematocrit margination ratio is 0.68 to 0.90. Two model platelet concentration profiles are fit to the experimental results.     

Finite platelet size could be responsible for platelet margination effect

Blood flows through vessels as a segregated suspension. Erythrocytes distribute closer to the vessel axis, whereas platelets accumulate near vessel walls. Directed platelet migration to the vessel walls promotes their hemostatic function. The mechanisms underlying this migration remain poorly understood, although various hypotheses have been proposed to explain this phenomenon (e.g., the available volume model and the drift-flux model). To study this issue, we constructed a mathematical model that predicts the platelet distribution profile across the flow in the presence of erythrocytes. This model considers platelet and erythrocyte dimensions and assumes an even platelet distribution between erythrocytes. The model predictions agree with available experimental data for near-wall layer margination using platelets and platelet-modeling particles and the lateral migration rate for these particles. Our analysis shows that the strong expulsion of the platelets from the core to the periphery of the blood vessel may mainly arise from the finite size of the platelets, which impedes their positioning in between the densely packed erythrocytes in the core. This result provides what we believe is a new insight into the rheological control of platelet hemostasis by erythrocytes.     

A shear-restricted pathway of platelet procoagulant activity is regulated by IQGAP1

Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to approximately 2.5 times the normal shear stress, or approximately 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation (cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca(2+)](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.     

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression.

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the "in vitro" ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.     

Plant food delphinidin-3-glucoside significantly inhibits platelet activation and thrombosis: novel protective roles against cardiovascular diseases

Delphinidin-3-glucoside (Dp-3-g) is one of the predominant bioactive compounds of anthocyanins in many plant foods. Although several anthocyanin compounds have been reported to be protective against cardiovascular diseases (CVDs), the direct effect of anthocyanins on platelets, the key players in atherothrombosis, has not been studied. The roles of Dp-3-g in platelet function are completely unknown. The present study investigated the effects of Dp-3-g on platelet activation and several thrombosis models in vitro and in vivo. We found that Dp-3-g significantly inhibited human and murine platelet aggregation in both platelet-rich plasma and purified platelets. It also markedly reduced thrombus growth in human and murine blood in perfusion chambers at both low and high shear rates. Using intravital microscopy, we observed that Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for vessel occlusion. Dp-3-g also significantly inhibited thrombus growth in a carotid artery thrombosis model. To elucidate the mechanisms, we examined platelet activation markers via flow cytometry and found that Dp-3-g significantly inhibited the expression of P-selectin, CD63, CD40L, which reflect platelet α- and δ-granule release, and cytosol protein secretion, respectively. We further demonstrated that Dp-3-g downregulated the expression of active integrin αIIbβ3 on platelets, and attenuated fibrinogen binding to platelets following agonist treatment, without interfering with the direct interaction between fibrinogen and integrin αIIbβ3. We found that Dp-3-g reduced phosphorylation of adenosine monophosphate-activated protein kinase, which may contribute to the observed inhibitory effects on platelet activation. Thus, Dp-3-g significantly inhibits platelet activation and attenuates thrombus growth at both arterial and venous shear stresses, which likely contributes to its protective roles against thrombosis and CVDs.     

A novel mouse-driven ex vivo flow chamber for the study of leukocyte and platelet function

Various in vitro and in vivo techniques exist for study of the microcirculation. Whereas in vivo systems impress with their physiological fidelity, in vitro systems excel in the amount of reduction that can be achieved. Here we introduce the autoperfused ex vivo flow chamber designed to study murine leukocytes and platelets under well-defined hemodynamic conditions. In our model, the murine heart continuously drives the blood flow through the chamber, providing a wide range of physiological shear rates. We used a balance of force approach to quantify the prevailing forces at the chamber walls. Numerical simulations show the flow characteristics in the chamber based on a shear-thinning fluid model. We demonstrate specific rolling of wild-type leukocytes on immobilized P-selectin, abolished by a blocking MAb. When uncoated, the surfaces having a constant shear rate supported individual platelet rolling, whereas on areas showing a rapid drop in shear platelets interacted in previously unreported grapelike conglomerates, suggesting an influence of shear rate on the type of platelet interaction. In summary, the ex vivo chamber amounts to an external vessel connecting the arterial and venous systems of a live mouse. This method combines the strengths of existing in vivo and in vitro systems in the study of leukocyte and platelet function. autoperfused flow chamber; intravital microscopy; inflammation; thrombus formation     

Inhibition of 2-P-mercaptophenyl-1,4-naphthoquinone on human platelet function.

As widely assumed, platelets and coagulation system heavily influence the pathogenesis and progression of cardiovascular diseases. Some 1,4-naphthoquinone derivatives, such as vitamin K3, have been reported to increase the synthesis of coagulation proteins. In this study, we examine how 2-p-mercaptophenyl -1,4-naphthoquinone (NTP), a newly synthesized 1,4-naphthoquinone derivative, affects the platelet function in humans. A tapered parallel plate chamber which provided a range of shear stress covering the entire physiological range in human circulation is used to assess platelet adhesiveness on fibrinogen coated-surface. In addition, platelet aggregation and thromboxane B2 (TXB2) production by inducers are evaluated by the turbidimetric method and enzyme immunoassay kit, respectively. Moreover, platelets [Ca2+]i are measured using a dual-wavelength fluorescence spectrophotometer. Analysis results indicate that 1) NTP decreases the percentages of attached platelets at the locations in various shear stresses and the levels of platelet adhesiveness, denoted as the slope; 2) NTP can inhibit the platelet aggregation by ADP (2 microM) and collagen (25 microg/ml), and the IC50 are: 0.32 and 26.83 microg/ml, respectively; and 3) NTP markedly inhibits TXB2 formation and platelet [Ca2+]i elevation caused by ADP and collagen. Therefore, we conclude that NTP may inhibit platelet adhesiveness on fibrinogen coated-surface, aggregability, [Ca2+]i, and thromboxane production, and that it may be used as an antiplatelet agent.     

Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo.     

Tortuosity triggers platelet activation and thrombus formation in microvessels

Tortuous blood vessels are often seen in humans in association with thrombosis, atherosclerosis, hypertension, and aging. Vessel tortuosity can cause high fluid shear stress, likely promoting thrombosis. However, the underlying physical mechanisms and microscale processes are poorly understood. Accordingly, the objectives of this study were to develop and use a new computational approach to determine the effects of venule tortuosity and fluid velocity on thrombus initiation. The transport, collision, shear-induced activation, and receptor-ligand adhesion of individual platelets in thrombus formation were simulated using discrete element method. The shear-induced activation model assumed that a platelet became activated if it experienced a shear stress above a relative critical shear stress or if it contacted an activated platelet. Venules of various levels of tortuosity were simulated for a mean flow velocity of 0.10?cm s(-1), and a tortuous arteriole was simulated for a mean velocity of 0.47?cm s(-1). Our results showed that thrombus was initiated at inner walls in curved regions due to platelet activation in agreement with experimental studies. Increased venule tortuosity modified fluid flow to hasten thrombus initiation. Compared to the same sized venule, flow in the arteriole generated a higher amount of mural thrombi and platelet activation rate. The results suggest that the extent of tortuosity is an important factor in thrombus initiation in microvessels.     

Fluid shear as a possible mechanism for platelet diffusivity in flowing blood

Platelet transport theory is based on convection diffusion and describes adequately the influence of wall shear rate, platelet concentration and axial (down stream) position. Until now, the influence of the predominant factors affecting platelet adherence, the hematocrit and the red cell size, was not included in this theory. Their role remained hidden in the platelet diffusivity (Dw), which was assumed to be related to the shear rate (gamma) expressed in s-1 by a power law function Dw = m gamma n, in which m and n were thought to be constants. We have determined platelet diffusivity directly from platelet adherence to subendothelium as a function of axial distance in an in vitro perfusion system. Our results indicate that m is a constant with a value of (1.05 +/- 0.05) 10(-9) cm2 s-1 and that n is a function of the hematocrit (h) which is best approximated by a quadratic equation n = 0.297 + 1.29 h - 0.90 h2. The effect of red cell size was introduced by correcting the hematocrit containing factors in this quadratic equation for the square of the red cell diameter. This correction was made on the basis of theoretical considerations. The theoretically derived platelet adherence correlated closely with the previous experimental data regarding the effect of red cell size in which we found that the hemodynamic effect of red cells on platelet adherence decreases with decreasing red cell diameter.     

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb(alpha)-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.     

Peroxynitrite can affect platelet responses by inhibiting energy production

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 microM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3',5'-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 microM) resulted in decreased activities of NADH : ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.