Redundant mechanism of platelet adhesion to laminin and collagen under flow: involvement of von Willebrand factor and glycoprotein Ib-IX-V

Although the role of collagen in thrombosis has been extensively investigated, the contribution of other extracellular matrices is still unclear. We have recently reported that laminin stimulates platelet spreading through integrin alpha(6)beta(1)-dependent activation of the collagen receptor glycoprotein (GP) VI under static condition. Under physiological high and low shear conditions, platelets adhered to laminin, and this was strongly inhibited by an antibody that blocks association between GPIb-IX-V and von Willebrand factor (VWF). Moreover, platelets of type III von Willebrand disease or Bernard-Soulier syndrome adhered to laminin at a low shear condition but not at a high shear condition. The specific binding of laminin to VWF was confirmed by surface plasmin resonance spectroscopy (BIAcore). These findings suggest that laminin supports platelet adhesion depending on the interaction of VWF and GPIb-IX-V under pathophysiological high shear flow. This mechanism is similar to that of collagen. We propose that integrins, GPVI, GPIb-IX-V, and VWF represent a general paradigm for the interaction between platelets and subendothelial matrices.     

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.     

Increases in platelet and red cell counts,blood viscosity,and arterial pressure during mild surface cooling: factors in mortality from coronary and cerebral thrombosis in winter.

Six hours of mild surface cooling in moving air at 24 degrees C with little fall in core temperature (0.4 degree C) increased the packed cell volume by 7% and increased the platelet count and usually the mean platelet volume to produce a 15% increase in the fraction of plasma volume occupied by platelets. Little of these increases occurred in the first hour. Whole blood viscosity increased by 21%; plasma viscosity usually increased, and arterial pressure rose on average from 126/69 to 138/87 mm Hg. Plasma cholesterol concentration increased, in both high and low density lipoprotein fractions, but values of total lipoprotein and lipoprotein fractions were unchanged. The increases in platelets, red cells, and viscosity associated with normal thermoregulatory adjustments to mild surface cooling provide a probable explanation for rapid increases in coronary and cerebral thrombosis in cold weather. The raised arterial pressure and possibly cholesterol concentration may contribute to slower components of the increased thrombosis.     

An Investigation on Platelet Transport during Thrombus Formation at Micro-Scale Stenosis

This paper reports on an investigation of mass transport of blood cells at micro-scale stenosis where local strain-rate micro-gradients trigger platelet aggregation. Using a microfluidic flow focusing platform we investigate the blood flow streams that principally contribute to platelet aggregation under shear micro-gradient conditions. We demonstrate that relatively thin surface streams located at the channel wall are the primary contributor of platelets to the developing aggregate under shear gradient conditions. Furthermore we delineate a role for red blood cell hydrodynamic lift forces in driving enhanced advection of platelets to the stenosis wall and surface of developing aggregates. We show that this novel microfluidic platform can be effectively used to study the role of mass transport phenomena driving platelet recruitment and aggregate formation and believe that this approach will lead to a greater understanding of the mechanisms underlying shear-gradient dependent discoid platelet aggregation in the context of cardiovascular diseases such as acute coronary syndromes and ischemic stroke.     

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.     

The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor

Macromolecules and cells exposed to blood flow in the circulatory tree experience hydrodynamic forces that affect their structure and function. After introducing the general theory of the effects of shear forces on protein conformation, selected examples are presented in this review for biological macromolecules sensitive to shear stress. In particular, the biochemical effects of shear stress in controlling the von Willebrand Factor (VWF) conformation are extensively described. This protein, together with blood platelets, is the main actor of the early steps of primary haemostasis. Under the effect of shear forces > 30 dyn/cm², VWF unfolding occurs and the protein exhibits an extended chain conformation oriented in the general direction of the shear stress field. The stretched VWF conformation favors also a process of self aggregation, responsible for the formation of a spider web network, particularly efficient in the trapping process of flowing platelets. Thus, the effect of shear stress on conformational changes in VWF shows a close structure-function relationship in VWF for platelet adhesion and thrombus formation in arterial circulation, where high shear stress is present. The investigation of biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular interaction mechanisms involved in arterial thrombosis.     

Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.     

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.     

Model experiments on platelet adhesion in stagnation point flow

Experiments with glass models of arterial branchings and bends, perfused with bovine platelet rich plasma (PRP), revealed platelet deposition being strongly dependent on fluid dynamic factors. Predilection sites of platelet deposits are characterized by flow vectors directed against the wall, so-called stagnation point flow. Thus collision of suspended particles with the wall, an absolute prerequisite for adhesion of platelets to surfaces even as thrombogenic as glass, appears mediated by convective forces. The extent of platelet deposition is correlated to the magnitude of flow components normal to the surface as well as to the state of biological activation of the platelets. The latter could be effective by an increase in hydrodynamically effective volume, invariably associated with the platelet shape change reaction to biochemical stimulants like ADP. The effect of altered rheological properties of platelets upon their deposition and of mechanical properties of surfaces was examined in a stagnation point flow chamber. Roughnesses in the order of 5 microns, probably by creating local flow disturbances, significantly enhance platelet adhesion, as compared to a smooth surface of identical chemical composition.     

Simulation of Platelet,Thrombus and Erythrocyte Hydrodynamic Interactions in a 3D Arteriole with In Vivo Comparison

Cylindrical blood vessels, ellipsoid platelets and biconcave-shaped deformable erythrocytes (RBCs) are important participants in hemostasis and thrombosis. However, due to the challenge of combining these components in simulation tools, few simulation studies have included all of them in realistic three-dimensional models. In the present study, we apply a recently developed simulation model to incorporate these components and analyze the flow in a thrombotic tubular arteriole, particularly the detailed hydrodynamic interactions between the thrombus shape, RBCs and platelets. It was found that at certain azimuth positions, the velocity drops in the proximity of both the upstream and downstream edge of the thrombus, which is accompanied by a rapid velocity increase in the narrowed region. The RBCs alter the flow profiles significantly from the typical low Reynolds (Re) number flow, and also enhance the deposition of free flowing platelets onto the thrombus. By evaluating the platelet-thrombus interaction and platelet-RBC interaction together, several mechanisms of platelet deposition augmentation are identified. With in vivo data comparison, our model illustrates the potential of future thrombosis studies that incorporate detailed receptor-ligand adhesion modules.     

The clinical significance of whole blood viscosity in (cardio)vascular medicine

Whole blood is a non-Newtonian fluid, which means that its viscosity depends on shear rate. At low shear, blood cells aggregate, which induces a sharp increase in viscosity, whereas at higher shear blood cells disaggregate, deform and align in the direction of flow. Other important determinants of blood viscosity are the haematocrit, the presence of macro-molecules in the medium, temperature and, especially at high shear, the deformability of red blood cells. At the sites of severe atherosclerotic obstructions or at vasospastic locations, when change of vessel diameter is limited, blood viscosity contributes to stenotic resistance thereby jeopardising tissue perfusion. However, blood viscosity plays its most important role in the microcirculation where it contributes significantly to peripheral resistance and may cause sludging in the postcapillary venules. Apart from the direct haemodynamic significance, an increase in blood viscosity at low shear by red blood cell aggregation is also associated with increased thrombotic risk, as has been demonstrated in atrial fibrillation. Furthermore, as increased red blood cell aggregation is a reflection of inflammation, hyperviscosity has been shown to be a marker of inflammatory activity. Thus, because of its potential role in haemodynamics, thrombosis and inflammation, determination of whole blood viscosity could provide useful information for diagnostics and therapy of (cardio)vascular disease.     

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.     

Platelet deposition onto fibrin-coated surfaces under flow conditions

Platelet deposition on fibrin-coated surfaces and release from these adherent platelets were studied in an in vitro flow system. When a mixed suspension of washed platelets and red cells flowed through a fibrin-coated glass tube, only platelets were deposited onto the fibrin-coated surfaces. The density of adhered platelets increased with flow time and decreased with distance from the tube inlet. The adhesion rate increased with increasing shear rates from 45 s-1 to 180 s-1. This adhesion process appears to fit a diffusion-limited mathematical model. Comparing with glass and other protein-coated surfaces such as collagen, fibrinogen, or albumin coated surfaces, the number of adhered platelet per unit area decreased in the following descending order: collagen, fibrin, fibrinogen, glass, albumin. On the other hand, the degree of release reaction from these platelets decreased by another order: collagen, glass, fibrinogen, fibrin. We observed little release from platelets that were in contact with a fibrin-coated surface. Our results suggest that platelets specifically adhere to fibrin-coated surface and that this interaction does not induce platelet release.     

Fluid shear regulates the kinetics and receptor specificity of Staphylococcus aureus binding to activated platelets

The interaction between surface components on the invading pathogen and host cells such as platelets plays a key role in the regulation of endovascular infections. However, the mechanisms mediating Staphylococcus aureus binding to platelets under shear remain largely unknown. This study was designed to investigate the kinetics and molecular requirements of platelet-S. aureus interactions in bulk suspensions subjected to a uniform shear field. Hydrodynamic shear-induced collisions augment platelet-S. aureus binding, which is further potentiated by platelet activation with stromal derived factor-1beta. Peak adhesion efficiency occurs at low shear (100 s(-1)) and decreases with increasing shear. The molecular interaction of platelet alpha(IIb)beta(3) with bacterial clumping factor A through fibrinogen bridging is necessary for stable bacterial binding to activated platelets under shear. Although this pathway is sufficient at low shear (相似文献   

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 lysis and aggregation in shear fields.

A rotational viscometer was used to study the effects of shear stress on platelets in human platelet-rich plasma (PRP). For 5-min exposure times, shear stresses above 160 dynes/cm2 induced platelet lysis (as determined by release of platelet lactic dehydrogenase). For 30-s exposure times, shear stresses greater than 600 dynes/cm2 were required to induce platelet lysis. The platelet counts of sheared PRP were decreased to as low as one-fifth the original count due largely to shear-induced aggregation. The count is a minimum at intermediate stress levels (200-400 dynes/cm2). Higher stresses induce disaggregation as well as lysis. The diminution in the counts was partially reversed in 2 h incubation after cessation of shearing. Experiments were carried out with three different viscometer configurations so that the shear stress and the solid surface area access could be varied independently. Surface access was not a significant variable in the conditions of the experiments. Thus aggregation and lysis may be induced by stress effects alone as well as by solid surface effects. The results also show that the response of platelets to shear stress is strongly dependent on exposure time. Platelets are much less resistant to shear stress than red cells for relatively long exposure times. However, the converse is true for very short exposure times.     

Ristocetin-induced self-aggregation of von Willebrand factor

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.     

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.