Platelet kinetics in the pulmonary microcirculation in vivo assessed by intravital microscopy

Growing evidence supports the substantial pathophysiological impact of platelets on the development of acute lung injury. Methods for studying these cellular mechanisms in vivo are not present yet. The aim of this study was to develop a model enabling the quantitative analysis of platelet kinetics and platelet-endothelium interaction within consecutive segments of the pulmonary microcirculation in vivo. New Zealand White rabbits were anesthetized and ventilated. Autologous platelets were separated from blood and labeled ex vivo with rhodamine 6G. After implantation of a thoracic window, microhemodynamics and kinetics of platelets were investigated by intravital microscopy. Velocities of red blood cells (RBCs) and platelets were measured in arterioles, capillaries and venules, and the number of platelets adhering to the microvascular endothelium was counted. Kinetics of unstimulated platelets was compared with kinetics of thrombin-activated platelets. Velocity of unstimulated platelets was comparable to RBC velocity in all vessel segments. Unstimulated platelets passed the pulmonary microcirculation without substantial platelet-endothelial interaction. In contrast, velocity of activated platelets was decreased in all vascular segments indicating platelet margination and temporal platelet-endothelium interaction. Thrombin-activated platelets adhered to arteriolar endothelium; in capillaries and venules adherence of platelets was increased 8-fold and 13-fold, respectively. In conclusion, using intravital microscopy platelet kinetics were directly analyzed in the pulmonary microcirculation in vivo for the first time. In contrast to leukocytes, no substantial platelet-endothelium interaction occurs in the pulmonary microcirculation without any further stimulus. In response to platelet activation, molecular mechanisms enable adhesion of platelets in arterioles and venules as well as retention of platelets within capillaries. Copyright (C) 2002 S. Karger AG, Basel

Canine platelets express functional Toll-like receptor-4: lipopolysaccharide-triggered platelet activation is dependent on adenosine diphosphate and thromboxane A2 in dogs.

BackgroundFunctional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A2 (TxA2). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS).ResultsWe discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B2 secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA2 pathway.ConclusionExpression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA2 pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis

Human platelets repurposed as vehicles for in vivo imaging of myeloma xenotransplants.

Human platelets were identified in tumors by Trousseau in 1865, although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers. In this study we exploit and enhance platelet interactions in tumor microenvironments by introducing tumor-targeting and imaging functions. The first step in repurposing human platelets as vehicles for tumor-targeting was to inhibit platelet-aggregation by cytoplasmic-loading of kabiramide (KabC), a potent inhibitor of actin polymerization and membrane protrusion. KabC-Platelets can accumulate high levels of other membrane-permeable cytoxins and probes, including epidoxorubicin, carboxyfluorescein di-ester and chlorin-e6. Finally, mild reaction conditions were developed to couple tumor-targeting proteins and antibodies to KabC-platelets. Fluorescence microscopy studies showed KabC-platelets, surface-coupled with transferrin and Cy5, bind specifically to RPMI8226 and K562 cells, both of which over-express the transferrin receptor. Repurposed platelets circulate for upto 9-days a feature that increases their chance of interacting with target cells. KabC-platelets, surface-coupled with transferrin and Cy7, or chlorin-e6, and injected in immuno-compromised mice were shown to accumulate specifically in sub-cutaneous and intra-cranial myeloma xenotransplants. The high-contrast, in vivo fluorescence images recorded from repurposed platelets within early-stage myeloma is a consequence in part of their large size (φ~2µm), which allows them to transport 100 to 1000-times more targeting-protein and probe molecules respectively. Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help stage tumor environments for an immunotherapy, or with combinations of therapeutic antibodies and therapeutic agents to target and treat cardiovascular and neurologic diseases

Role of P-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

Background: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. Methods: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. Results: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. Conclusion: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction. Copyright (c) 2006 S. Karger AG, Basel

An in vitro study of the adhesion of blood platelets onto vascular catheters. Part I

The adhesion of human blood platelets onto vascular catheters was studied using a specially designed perfusion chamber. Polyurethane catheters were exposed to citrated human blood for different periods (up to 20 min) and at different wall shear rates (190, 260, 330 sec-1). The rate of platelet adhesion was determined using 111In-labeled platelets, while the morphology of adhering platelets was investigated using scanning electron microscopy. A linear increase in platelet adhesion was found within the first 10 min of perfusion, after which a plateau value was reached. The number of adhering platelets did not vary significantly with the shear rates applied, which may indicate that within the range of shear rates studied, the adhesion of platelets onto the catheter surface is mainly determined by the rate of the reaction between the platelets and the material surface. Catheters coated with a conjugate of heparin and albumin showed a four- to five-fold reduction in platelet adhesion as compared to uncoated catheters. This reduction in platelet adhesion was not only due to the presence of albumin moieties at the surface but also to the presence of heparin residues in the adsorbed albumin-heparin conjugate

Platelet Biochemistry and Morphology after Cryopreservation

Platelet cryopreservation has been investigated for several decades as an alternative to room temperature storage of platelet concentrates. The use of dimethylsulfoxide as a cryoprotectant has improved platelet storage and cryopreserved concentrates can be kept at −80 °C for two years. Cryopreserved platelets can serve as emergency backup to support stock crises or to disburden difficult logistic areas like rural or military regions. Cryopreservation significantly influences platelet morphology, decreases platelet activation and severely abrogates platelet aggregation. Recent data indicate that cryopreserved platelets have a procoagulant phenotype because thrombin and fibrin formation kicks in earlier compared to room temperature stored platelets. This happens both in static and hydrodynamic conditions. In a clinical setting, low 1-h post transfusion recoveries of cryopreserved platelets represent fast clearance from circulation which may be explained by changes to the platelet GPIbα receptor. Cryopreservation splits the concentrate in two platelet subpopulations depending on GPIbα expression levels. Further research is needed to unravel its physiological importance. Proving clinical efficacy of cryopreserved platelets is difficult because of the heterogeneity of indications and the ambiguity of outcome measures. The procoagulant character of cryopreserved platelets has increased interest for use in trauma stressing the need for double-blinded randomized clinical trials in actively bleeding patients

A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation.

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation

RGS10 shapes the hemostatic response to injury through its differential effects on intracellular signaling by platelet agonists.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5\u27-diphosphate (ADP) and thromboxane A2 (TxA2). Hemostatic thrombi formed in RGS10-/- mice were larger than in controls, with the increase due to expansion of the shell but not the core. Clot retraction was slower, and average packing density was reduced. Deleting RGS10 had agonist-specific effects on signaling. There was a leftward shift in the dose/response curve for the thrombin receptor (PAR4) agonist peptide AYPGKF but no increase in the maximum response. This contrasted with ADP and TxA2, both of which evoked considerably greater maximum responses in RGS10-/- platelets with enhanced Gq- and Gi-mediated signaling. Shape change, which is G13-mediated, was unaffected. Finally, we found that free RGS10 levels in platelets are actively regulated. In resting platelets, RGS10 was bound to 2 scaffold proteins: spinophilin and 14-3-3γ. Platelet activation caused an increase in free RGS10, as did the endothelium-derived platelet antagonist prostacyclin. Collectively, these observations show that RGS10 serves as an actively regulated node on the platelet signaling network, helping to produce smaller and more densely packed hemostatic thrombi with a greater proportion of fully activated platelets

Bulk and wetting phenomena in a colloidal mixture of hard spheres and platelets

Density functional theory is used to study binary colloidal fluids consisting of hard spheres and thin platelets in their bulk and near a planar hard wall. This system exhibits liquid-liquid coexistence of a phase that is rich in spheres (poor in platelets) and a phase that is poor in spheres (rich in platelets). For the mixture near a planar hard wall, we find that the phase rich in spheres wets the wall completely upon approaching the liquid demixing binodal from the sphere-poor phase, provided the concentration of the platelets is smaller than a threshold value which marks a first-order wetting transition at coexistence. No layering transitions are found in contrast to recent studies on binary mixtures of spheres and non-adsorbing polymers or thin hard rods.Comment: 6 pages, 4 figure