Cellular pathology of atherosclerosis: smooth muscle cells promote adhesion of platelets to cocultured endothelial cells

Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1. Recapitulating the model using EC alone, we found that a combination of TGF-beta1 and TNF-alpha promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or alpha(IIb)beta3 integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF-alpha and TGF-beta1 revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 s(-1). Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.     

Porcine platelets in vitro and in vivo studies: relevance to human thrombosis research.

This review summarizes present knowledge on porcine platelets in vitro and recent studies on in vivo activation of platelets in the pig. There are certain differences compared to human platelets: Platelet aggregation and secretion cannot be achieved by epinephrine, and the arachidonate pathway seems poorly developed in porcine platelets. Genetic models for von Willebrand disease (vWD) and storage pool deficiency (SPD) have been developed in the pig. Several models for the study of in vivo platelet deposition and early thrombus formation have been developed. Platelet radio-labeling techniques (with 111In) have been used extensively. We conclude that the pig seems to be a good choice for the investigation of in vivo platelet activation and deposition based on present knowledge of porcine platelets and on already established animal models.     

Small cells,big effects: the role of platelets in transplant vasculopathy

Platelets are the cellular mediator of thrombosis, but also represent an important part of the immune system. Platelets store numerous pre-formed inflammatory molecules in their granules and produce immune mediators de novo upon stimulation. Activated platelets express adhesion molecules that can interact with endothelial cells and leukocytes both at the site of inflammatory insult and in the circulation. Platelets therefore drive and potentiate vascular inflammation by direct and indirect interactions with both endothelial cells and leukocytes. Platelet activation has been associated with the pathogenesis of inflammatory diseases such as atherosclerosis, arthritis, and cerebral malaria, in addition to transplant vasculopathy. In this review we will discuss immune roles for platelets in the context of transplant vascular inflammation.     

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.     

Studies of fibrinogen binding to porcine platelets by flow cytometry: a method for studies of porcine platelet activation

Platelets have a central function in haemostasis. They also participate in arterial thrombus formation in vascular disorders. Platelets have an important role in initiating and mediating ischaemia and related complications of ischemic heart disease. Several research groups are thus studying platelet activation and developing new platelet inhibitors. Platelet function is dependent upon membrane receptors and their interaction with other proteins. Binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor is a prerequisite for platelet aggregation and thrombus formation. Thus, several GPIIb/IIIa inhibitors have been developed of which abciximab is the clinically most widely used. Pigs are often used for experimental studies. We have developed a flow cytometry assay for measuring porcine platelet activation utilising an FITC-labelled chicken anti-fibrinogen antibody. ADP, ristocetin and thrombin induce fibrinogen binding to porcine platelets similarly to human platelets. Ristocetin induces platelet aggregation and microvesicle formation from porcine platelets as well as from human platelets.     

Platelet-endothelial cell interactions in murine antigen-induced arthritis

OBJECTIVES: Growing evidence supports the substantial pathophysiological impact of platelets on the development of rheumatoid arthritis. At present there are no methods for studying these cellular mechanisms in vivo. The aim of this study was to visualize and investigate platelet-endothelial cell interaction in the knee joint of mice with antigen-induced arthritis (AiA) by means of intravital microscopy. METHODS: In 14 mice (Balbc) intravital microscopic assessment was performed on day 8 after AiA induction in two groups (controls, AiA). The severity of AiA was assessed by measuring knee joint swelling and by histological scoring. Ex vivo fluorescently labelled rolling and adherent platelets and leucocyte-endothelium interactions were investigated by intravital fluorescence microscopy. RESULTS: Swelling of the knee joint as well as histological score was significantly enhanced in arthritic animals compared with controls. In control mice intravital microscopy revealed low baseline rolling and sticking of leucocytes and fluorescently labelled platelets. AiA induced a significant increase in the fraction of rolling leucocytes (3 times) and rolling platelets (6 times) compared to the control group. Furthermore, AiA induction resulted in a significantly enhanced number of adherent leucocytes (3-fold) and adherent platelets (12-fold) in comparison with control animals. CONCLUSIONS: Platelet kinetics were directly analysed using intravital microscopy in the arthritic microcirculation in vivo for the first time. We provide the first evidence that platelets accumulate in arthritic vessels, indicating platelet activation due to AiA. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of rheumatoid arthritis.     

Thrombus formation without platelets under inflammatory condition: an in vitro study

Platelet derived microvesicles, which are shed from platelets upon platelet activation, interact with monocytes in the blood. In this study the nature of this interaction was characterized in a model system with the monocytic cell line MM6 and isolated platelet derived microvesicles (PMV). The interaction of PMV with MM6 is separated in two consecutive steps, which are partially overlapped in time. In a first step there is an immediate conjugate formation with single MM6 and PMV, which was proved microscopically and by cytometry measurements. This process is dependent on CD62P, determined by an inhibition after pre-incubation with anti-CD62P. After a lag time of 4 min this process is supplemented by an aggregate formation of single conjugates, which leads finally to one macroscopic visible aggregate. The Nature of this aggregate was characterized by immunohistochemistry and laser aggregatometry. An addition of GPRP blocks the formation of a fibrin network and also the aggregate formation, proving the necessity of fibrin network formation. This was also shown by diminishing the aggregate formation by addition of hirudin. Finally fluorescent microscopic images proved the necessity of a fibrin network holding MM6 cell/PMV aggregates together. Even pure PMV can form such an aggregate only visible as thin film and less stable as the cell PMV aggregate. The described process might be important in vivo causing thrombotic events without direct involvement of platelets. Especially in situations with extreme PMV levels, such as acute coronary heart disease, trauma and sepsis, these events could lead to the appearance of haemostatic complications.     

Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 > thrombin plus collagen > collagen > thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.     

Glycoprotein IIb and IIIa retention on fibrinogen-coated surfaces after lysis of adherent platelets

The platelet-membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex is essential for platelet aggregation and is involved in the attachment of platelets to thrombogenic surfaces. This study shows the retention of GPIIb and GPIIIa on immobilized fibrinogen after Triton X-100 (Sigma Chemical Co, St Louis, MO) lysis of adherent platelets. Glycoproteins were detected using subunit specific monoclonal antibodies in a modified enzyme-linked immunosorbent assay procedure. GPIIb-IIIa retention was judged to be specific relative to GPIb recovery, and was modulated by platelet activation. Platelet exposure to adenosine diphosphate or thrombin, but not A23187 or chymotrypsin, markedly enhanced GPIIb and GPIIIa recovery relative to that observed with unstimulated platelets, or prostaglandin E1-treated platelets. Moreover, lysis of adherent platelets in the presence of 10 mmol/L EDTA, under conditions promoting GPIIb-IIIa complex dissociation (pH 8.1, 60 minutes, 37 degrees C), had no effect on GPIIb or GPIIIa subunit recovery. Platelet activation with Zn+2 also enhanced GPIIb and GPIIIa recovery on fibrinogen-coated surfaces over that observed with unstimulated platelets, but GPIIb and IIIa retention was EDTA sensitive. This correlated with the EDTA-reversible nature of Zn+2- activated platelet adhesion to fibrinogen-coated surfaces. The data (1) show that platelet adhesion to fibrinogen is accompanied by the induction of high-affinity interactions between GPIIb-IIIa and immobilized fibrinogen that are EDTA-resistant and enhanced by platelet activation with some but not all agonists, and (2) implicate these interactions in stabilizing platelet contacts with fibrinogen-coated surfaces.     

Platelet heterogeneity based on lactic dehydrogenase activity

Mean lactic dehydrogenase (LDH) activity of human platelets is about 5 nU/platelet; expressed on a cell volume basis, it is 0.5 nU/fl. Platelet separation by means of centrifugation on a Percoll discontinuous gradient gives different subpopulations with a clear-cut change of LDH at a mean platelet volume (MPV) of about 6 fl. The LDH content of platelets with a MPV > 6 fl is constant (about 0.5 nU/fl), while that of small platelets (3 < MPV < 6 fl) is inversely correlated with the MPV and reaches a value of 1.5-2.0 nU/fl. Because small platelets are granule-depleted, we suggest that platelet LDH activity be considered as an in vivo activation index.     

Thrombus resistance to lysis and reocclusion after thrombolysis: the role of platelets.

Thrombolytic therapy reduces the mortality rate in patients with acute myocardial infarction. However, thrombolytic agents, which are primarily directed toward the lysis of fibrin, fail to achieve reperfusion in at least 20% of patients and an early reocclusion occurs in up to 25% of cases. The role of platelets in both thrombus resistance to lysis and early reocclusion is demonstrated by experimental and clinical observations. Platelets and the fibrinolytic system interact in numerous ways. Platelet activation can potentiate or inhibit the fibrinolytic process. The activation of the fibrinolytic system has been found to induce either an activation or an inhibition of platelet function. In animal models it has been shown that platelet-rich thrombi are resistant to lysis, and that platelets and fibrin still accumulate on lysing thrombi during effective thrombolysis. One of the most attractive strategies to optimize thrombolytic therapy is adjunctive treatment with pharmacological agents which inhibit platelet function.     

Impaired platelet function correlates with multi-organ dysfunction. A study of patients with sepsis

Patients with sepsis often suffer from haemostatic disturbances such as haemorrhage and disseminated intravascular coagulation (DIC). Considering the pivotal role of platelets in haemostasis, we have investigated platelet function by flow cytometry in 16 patients with sepsis for a better understanding of their haemostatic function. We have also investigated whether platelet function correlates with the severity of disease assessed by multiple organ dysfunction (MOD) score and patient outcome. The platelet response ex vivo after stimulation with agonists, measured as platelet fibrinogen, binding was low in comparison with healthy volunteers ( n = 30). This could reflect a previous response to agonists in vivo , which lead to platelet activation and consumption and formation of microthrombi that could then participate in the development of M OD. The platelets that remain in the circulation might be the result of a selection process where the most active platelets have already been consumed, and the remaining population consists of less active platelets. Another explanation might be desensitization of the remaining platelets because of exposure to agonists in vivo . Platelet activation with the agonists ADP and arachidonic acid were predictive of subsequent development of MOD and final patient outcome.     

Activation of alphaIIbbeta3 is a sufficient but also an imperative prerequisite for activation of alpha2beta1 on platelets

Platelet integrins alpha2beta1 and alphaIIbbeta3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin alphaIIbbeta3 is not only sufficient, but also a prerequisite for alpha2beta1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of alpha2beta1, as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of alphaIIbbeta3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful alpha2beta1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of alphaIIbbeta3 for proper alpha2beta1 activation on platelets was demonstrated by using the alphaIIbbeta3 antagonist aggrastat, which was able to completely abolish alpha2beta1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking alphaIIbbeta3 confirmed the indispensability of alphaIIbbeta3 for alpha2beta1 activation.     

Flow cytometric analysis of platelet activation under calcium ion-chelating conditions

Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA.     

Platelet-neutrophil interactions. Possible relevance in the pathogenesis of thrombosis and inflammation.

Evidence indicates that complex interactions occur between platelets and neutrophils. Mediators released by activated platelets may act as stimuli for neutrophils, but platelets may also limit neutrophil activation. Similarly, neutrophils, depending upon their degree of activation, may bring about both enhancement and inhibition of platelet responses. The elaborate cross-talk among platelets and neutrophils, cells involved in hemostatic and phlogistic reactions, may condition the development and the course of thrombotic and inflammatory diseases; therefore, modulation of platelet-neutrophil interactions may represent a therapeutic target in these conditions. In this review, the mediators involved in the platelet-dependent modulation of neutrophil activation and, conversely, the mechanisms whereby neutrophils modify platelet responses are described. Moreover, the possible relevance of these interactions in the pathogenesis of thrombosis and inflammation is discussed.     

Platelet activation in hemolytic uremic syndrome

Platelet consumption in platelet-fibrin aggregates leading to thrombocytopenia and small vessel obstruction are major features of the hemolytic uremic syndrome (HUS). Although thrombocytopenia has been correlated to poor prognosis, the mechanisms by which thrombocytopenia develops in HUS have not been completely elucidated. However, plausible explanations have been platelet contact with thrombogenic surfaces and/or direct contact with an aggregating agent. This article summarizes several mechanisms of platelet activation, interactions with leukocytes, chemokine release, complement activation, and antimicrobial defense. Specific mechanisms are outlined by which platelets may be activated, leading to thrombocytopenia during HUS. In diarrhea-associated HUS Shiga toxin has been shown to injure the endothelium, thus exposing the subendothelium, releasing tissue factor, and rendering the vessel wall prothrombotic. Shiga toxin also binds to and activates platelets. The toxin may activate endothelial cells and platelets simultaneously. In atypical HUS the alternative complement pathway is activated because of mutations in complement regulatory proteins. Mutated factor H does not bind to endothelium and platelets efficiently, enabling complement activation on these cells. In thrombotic thrombocytopenic purpura, intravascular platelet clotting occurs due to dysfunction of the von Willebrand factor (VWF) -cleaving protease ADAMTS13. Thrombi are formed by binding of platelets to ultralarge VWF multimers.     

Collagen-induced platelet activation

Platelet collagen receptors, such as Gp VI, are attractive targets for antithrombotic therapy. In this paper, I discuss the current knowledge regarding collagen-platelet interactions, including the role of platelet receptors, the recognition of collagen by platelets, the effect of the interaction on platelet activation and thrombosis and the effect of collagen structure on the platelet interaction, and highlight the areas in which additional information is required to pursue the goal of antithrombotic therapy, using the collagen-platelet interaction as the site of intervention. Understanding the detail of the receptor recognition motifs within collagen not only may reveal new antithrombotic targets within collagen, but will almost certainly lead to the development of defined reagents that can be used in vitro and ex vivo to explore thrombus formation further.     

Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.     

Platelet lipoprotein interplay: trigger of foam cell formation and driver of atherosclerosis

In the last decade, it was recognized that platelets and lipoproteins play a pivotal role in both early and late atherogenesis. Beside cellular interactions of platelets with other blood cells and vascular cells, interactions with lipoproteins seem to be quite important. Lipoproteins are fundamental 'players' in atherogenesis since they change the properties of different cells involved in atherosclerosis and thrombosis. Several studies have already shown that low density lipoproteins (LDL) are involved in the initiation of platelet signalling pathways. Platelets of hypercholesterolemic patients show hyperaggregability in vitro and enhanced activity in vivo. This review elucidates the major aspects concerning how native and modified lipoproteins influence the activation and metabolic behaviour of platelets, and shows a new way by which platelet-mediated lipoprotein transfer might contribute to foam cell formation. In hyperlipidaemia, circulating platelets are activated. This is accompanied by increased platelet aggregation, platelet-leukocyte aggregate formation, and platelet-induced superoxide anion production. Furthermore, oxidized LDL induces monocyte adhesion to the endothelium, migration and proliferation of smooth muscle cells, injures cells, interferes with nitric oxide release, and promotes procoagulant properties of vascular cells. New data about platelet-mediated lipoprotein transport and consequent foam cell formation, however, provide proof of how platelets might contribute to atheromatous lesion formation.