Control Platelet Activation Research Articles

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α(2A)-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Chapter 10 - Ectonucleotidases as Regulators of Purinergic Signaling in Thrombosis, Inflammation, and Immunity

Evolving studies in models of transplant rejection, inflammatory bowel disease, and cancer, among others, have implicated purinergic signaling in clinical manifestations of vascular injury and thrombophilia, inflammation, and immune disturbance. Within the vasculature, spatial and temporal expression of CD39 nucleoside triphosphate diphosphohydrolase (NTPDase) family members together with CD73 ecto-5'-nucleotidase control platelet activation, thrombus size, and stability. This is achieved by closely regulated phosphohydrolytic activities to scavenge extracellular nucleotides, maintain P2-receptor integrity, and coordinate adenosinergic signaling responses. The CD38/CD157 family of extracellular NADases degrades NAD(+) and generates Ca(2+)-active metabolites, including cyclic ADP ribose and ADP ribose. These mediators regulate leukocyte adhesion and chemotaxis. These mechanisms are crucial in vascular homeostasis, hemostasis, thrombogenesis, and during inflammation. There has been recent interest in ectonucleotidase expression by immune cells. CD39 expression identifies Langerhans-type dendritic cells and efficiently distinguishes T regulatory cells from other resting or activated T cells. CD39, together with CD73 in mice, serves as an integral component of the suppressive machinery of T cells. Purinergic responses also impact generation of T helper-type 17 cells. Further, CD38 and changes in NAD(+) availability modulate ADP ribosylation of the cytolytic P2X7 receptor that deletes T regulatory cells. Expression of CD39, CD73, and CD38 ectonucleotidases on either endothelial or immune cells allows for homeostatic integration and control of vascular inflammatory and immune cell reactions at sites of injury. Ongoing development of therapeutic strategies targeting these and other ectonucleotidases offers promise for the management of vascular thrombosis, disordered inflammation, and aberrant immune reactivity.

A Chemical Genetic Analysis of Platelet Activation Identifies An Antithrombotic Allosteric Modulator That Acts through Helix 8 of Par1

AbstractAbstract 483The essential challenge in pharmacologic control of platelet activation during thrombus formation is to achieve inhibition of platelet mediated thrombosis without impairing hemostasis. Inhibition of platelet GPCRs has focused primarily on inhibition of orthosteric binding sites, selecting for compounds that compete with endogenous ligands. However, small molecules that act at non-orthosteric sites can stabilize alternative GPCR conformations and modify GPCR function. Such allosteric modulators demonstrate saturable binding, selective effects on affinity and efficacy, and could provide improved physiologic control of platelet function. However, cell-based screens are required for the identification of such modulators. We screened a 16,000 compound library for inhibitors of platelet activation and identified a family of alkylated quinolines that modified Par1-mediated granule secretion. Activity of these compounds was determined by the length of their alkyl tails such that a two carbon addition to the tail converted a compound that inhibited Par1 activating peptide (SFLLRN)-induced P-selectin expression into a compound that augmented Par1-mediated activation. The most potent inhibitory compound, JF5 (IC50 = 4 μM), blocked Par1 activating peptide-induced GTPase activity and GTP[γ-35S] binding but not GTPγS-induced platelet activation in permeabilized platelets, thus indicating activity at Par1. The compound failed to inhibit platelet activation induced by a Par4 activating peptide, the thromboxane analog U46619, arginine vasopressin, PMA, or Ca2+ ionophore. Studies using cultured cells overexpressing specific GPCRs demonstrated that JF5 inhibited Par1-mediated activation, but failed to inhibit activation through TP, P2Y1 or EP3 receptors. However, JF5 did demonstrate inhibition of the α2A-adrenergic receptor. Radioligand binding studies confirmed non-competitive inhibition of the α2A-adrenergic receptor by JF5. Comparative phenotyping using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to select GPCRs. Evaluation of the amino acid sequence of these receptors demonstrated that sensitive receptors possessed a constrained 8th helix, as defined by a C-terminal palmitoylation site and interactions of the 8th helix with TM7 and the i1 loop. The 8th helix of GPCRs is oriented parallel to the intracellular face of the plasma membrane and is thought to transduce signals to cognate Gα subunits. To assess the hypothesis that the 8th helix contributes to the inhibitory activity of JF5, the compound was evaluated for its ability to inhibit IP3 generation in COS7 cells overexpressing wild-type Par1 or cells expressing a Par1 mutant in which residues 376–386 had been replaced with 3 alanine residues (ΔH8 Par1). JF5 completely inhibited SFLLRN-induced IP3 generation in cells expressing wild-type Par1. Inhibition was reversed in the ΔH8 Par1 mutant, confirming the involvement of the 8th helix in JF5 inhibitory activity. Despite its potent inhibition of Par1-mediated aggregation, secretion, and IP3 generation, JF5 did not inhibit SFLLRN-induced shape change. To evaluate the premise that JF5 inhibited coupling to Gαq but not Gα12/13, we determined the effect of JF5 on SFLLRN-induced Gα12-dependent decreased barrier function by measuring changes in transepithelial resistance (TER) in MDCK cells overexpressing Gα12. JF5 demonstrated no inhibition of SFLLRN-mediated decrease in TER at concentrations up to 200 μM, whereas the orthosteric Par1 inhibitor, Sch79797, completely blocked SFLLRN-induced TER at 1 μM. These results confirmed that JF5 inhibits Par1 coupling to Gαq, but not Gα12. JF was also evaluated in a mouse model of thrombus formation following laser-induced injury of cremaster arterioles. JF5 inhibited thrombus formation with an IC50 of 1 mg/kg and delayed thrombus formation by 2.3-fold. Evaluation of the 8th helix of mouse Par4 demonstrated that, unlike the 8th helix of human Par4, it contained a palmitoylation site and is predicted to have a constrained conformation based on interactions with TM7. These results indicate a role for the 8th helix in conferring sensitivity to small molecules and show that this sensitivity can be exploited to control platelet activation during thrombus formation. JF5 will serve as a useful probe to evaluate the role of the 8th helix in coupling of GPCRs to cognate Gα subunits. Disclosures:No relevant conflicts of interest to declare.

Targeting Platelet G-Protein Coupled Receptors (GPCRs): Looking Beyond Conventional GPCR Antagonism

Platelets are key mediators of thrombosis. Drugs that interfere with platelet activation substantially improve survival in arterial thrombotic disease. One attractive family of drug targets which has already been exploited in the development of antiplatelet agents are G-protein coupled receptors (GPCRs). Yet limitations of present antiplatelet agents, including incomplete efficacy, bleeding and drug resistance, have spurred the development of new drugs directed at alternative platelet GPCRs. Compounds that target platelet receptors including P2Y(12), protease-activated receptor-1 (PAR1), the thromboxane A(2) receptor (TP receptor), the prostaglandin receptor (EP3 receptor), and the serotonin receptor (5-HT(2A) receptor) have been identified and are in various phases of clinical development and use. Yet, improving the clinical profile of reagents targeting platelet GPCRs may not be only a matter of blocking alternative GPCRs. A more detailed understanding of GPCR function is leading to the development of pharmacophores with novel mechanisms of drug action. Identifying compounds that work by alternative mechanisms may be equally or more valuable than developing reagents targeting different GPCRs. Pharmacological concepts such as GPCR oligomerization, allosterism, and targeting GPCR-G protein interactions may ultimately provide opportunities to more effectively control platelet activation in the clinical setting. In this review, we will discuss the utility and limitations of GPCR-targeted antiplatelet therapies in clinical use and development. We will also consider emerging concepts in GPCR pharmacology that have the potential to foster the development of GPCR-targeted reagents with novel mechanisms of action and improved clinical profiles.

The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O 2 −) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO −) that is formed from the reaction between O 2 − and nitric oxide (NO). There are conflicting data on ONOO − effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O 2 −, yielding ONOO −. Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 × 10 8 platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001–100 μM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O 2 − and ONOO − scavengers superoxide dismutase (SOD) and -(−)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human α-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O 2 − generation. Superoxide anion signaling pathway includes ONOO − formation and α-actinin nitration.

2008 Russell Ross Memorial Lectureship in Vascular Biology—Activating and Inhibitory Receptors in Blood and Vascular Cells

The activation state of circulating blood cells is a tightly controlled process that is continuously influenced by both stimulatory and inhibitory events, and proper integration of the signals transduced by physical and chemical mediators to which cells are exposed is crucial for maintaining homeostasis. Studies carried out over the past 20 years has led to the identification of numerous plasma membrane receptors linked to intracellular signaling pathways that regulate cellular activation, signal transmission, and later, adaptation. Human platelets are both a valuable and clinically relevant model system in which to study such activating and inhibitory processes at the molecular level, as their dysregulation can lead to bleeding, inflammation, thrombosis, and cardiovascular disease. Platelets express G protein-coupled receptors, kinase- and phosphatase-linked receptors, and a host of adhesion molecules, and there is growing recognition that these are intricately intertwined, making rational intervention to control platelet activation and platelet aggregation an increasingly approachable, albeit challenging, possibility. This lecture will highlight our recent understanding of the complex interplay between immunoreceptor tyrosine-based activation motif (ITAM)–bearing and immunoreceptor tyrosine-based inhibitory motif (ITIM)–bearing receptors that exists in platelets and other vascular cells, with an emphasis on the role that such receptors play in human health and the treatment of cardiovascular disease.

Spatially distinct production of reactive oxygen species regulates platelet activation

Platelets play a key role in hemostasis and changes in redox balance are known to alter platelet activation and aggregation. Interestingly, activation of platelets leads to production of reactive oxygen species (ROS), but the role(s) of these ROS remain unclear. Using flow cytometry and chemiluminescence, agonist-induced ROS generation was found to be spatially distinct with stimulation through the major collagen receptor GPVI inducing only intraplatelet ROS while thrombin induced production of extracellular ROS. Platelet activation by either the GPVI-selective agonist convulxin or thrombin was differentially regulated by ROS generation. Thus, surface expression of CD62P, CD40L, or activated integrin αIIbβ3 was abrogated by pharmacologic antioxidants but externalization of phosphatidylserine was not inhibited. Furthermore, extracellular antioxidants SOD/catalase markedly inhibited thrombin-, but not convulxin-, induced CD62P expression and αIIbβ3 activation. The data suggest that ROS selectively regulate biochemical steps in platelet activation and that distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation. Thus, targeting ROS with site-specific antioxidants may differentially regulate platelet activation via thrombin or collagen.

The immunoregulatory glycan‐binding protein galectin‐1 triggers human platelet activation

Platelet activation is a critical process during inflammation, thrombosis, and cancer. Here, we show that galectin-1, an endogenous lectin with immunoregulatory properties, plays a key role in human platelet activation and function. Galectin-1 binds to human platelets in a carbohydrate-dependent manner and synergizes with ADP or thrombin to induce platelet aggregation and ATP release. Furthermore, galectin-1 induces F-actin polymerization, up-regulation of P-selectin, and GPIIIa expression; promotes shedding of microvesicles; and triggers conformational changes in GPIIb/IIIa. In addition, exposure to this lectin favors the generation of leukocyte-platelet aggregates. A further mechanistic analysis revealed the involvement of Ca(2+) and cyclic nucleotide-dependent pathways in galectin-1-mediated control of platelet activation. Finally, expression of endogenous galectin-1 in human platelets contributes to ADP-induced aggregation. Our study reveals a novel unrecognized role for galectin-1 in the control of platelet physiology with potential implications in thrombosis, inflammation, and metastasis.

Programmed autologous cleavage of platelet receptors.

Platelet adhesion receptors play a critical role in vascular pathophysiology, and control platelet adhesion, activation and aggregation in hemostasis, thrombotic disease and atherogenesis. One of the key emerging mechanisms for regulating platelet function is the programmed autologous cleavage of platelet receptors. Induced by ligand binding or platelet activation, proteolysis at extracellular (ectodomain shedding) or intracellular (cytoplasmic domain deactivation) sites down-regulates the adheso-signaling function of receptors, thereby controlling not only platelet responsiveness, but in the case of ectodomain shedding, liberating soluble ectodomain fragments into plasma where they constitute potential modulators or markers. This review discusses the underlying mechanisms for dual proteolytic pathways of receptor regulation, and the impact of these pathways on thrombus formation and stability in vivo.

Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets

Extracellular nucleotide P2-receptor-mediated effects on platelets, leukocytes and endothelium are modulated by ecto-nucleotidases. These ecto-enzymes hydrolyze extracellular nucleotides to the respective nucleosides. The dominant ecto-nucleotidase expressed by the endothelium, by monocytes and vascular smooth muscle cells is CD39/NTPDase1. Ecto-nucleotidase biochemical activity of CD39 is lost at sites of acute vascular injury, such as in ischemia reperfusion and immune graft rejection. CD39L(Like)1/NTPDase2, a related protein, is associated with the basolateral surface of endothelium, the adventitia of vessels and microvascular pericytes. CD39/NTPDase1 hydrolyzes both tri- and diphosphonucleosides and blocks platelet aggregation responses to ADP. In contrast, CD39L1/NTPDase2, a preferential nucleoside triphosphatase, activates platelets by preferentially converting ATP to ADP, the major agonist of platelet P2 receptors. Spatial and temporal expression of NTPDases in the vasculature appears to control platelet activation, thrombus size and stability by regulating phosphohydrolytic activity and consequent P2 receptor signaling. Constitutively circulating microparticles appear to be associated with functional NTPDases, and accumulation of these at sites of vascular injury might influence local thrombus formation and evolution. The phenotype of the cd39-null mouse is in keeping with disordered thromboregulation with heightened susceptibility to inflammatory vasculary reactions, increased permeability and high levels of tissue fibrin. Paradoxically, these mutant mice also exhibit a bleeding phenotype with differential platelet P2Y1 desensitization. Over-expression of CD39 at sites of vascular injury and inflammation by adenoviral vectors, by transgenesis or by the use of pharmacological modalities with soluble derivatives has been shown to have major potential in several animal models tested to date. Future clinical applications will involve the development of new therapeutic strategies to various inflammatory vascular diseases and in transplantation.

410. Adenovirus Activates Mouse Platelets and Induces Platelet Leucocyte Association

Adenovirus vectors have been used extensively in pre-clinical and clinical studies as agents for gene delivery. Acute, transient thrombocytopenia is a known adverse effect of adenovirus vector administration with a mechanism that currently remains unresolved. The fall in platelets appears 24 hours following IV administration of adenovector and is vector dose dependent. We have initiated experiments to investigate the mechanism of Ad induced thrombocytopenia. We examined the expression of the platelet activation marker P-selectin (CD-62p) on mouse platelets after incubation with adenovirus in vitro by means of flow cytometry. An indirect immunoflourescence approach was used with anti-mouse CD 62p monoclonal antibody and an FITC labeled rat antimouse-IgG. An appropriate negative isotype control and positive platelet activation control using thrombin were incorporated into these studies. The index of platelet activation (IPA), was used to quantify the activation effect of Ad on the platelets. This is the product of the mean channel fluorescence and the percentage of positive cells. We also examined Platelet leucocyte association (PLA) using the direct immunoflourescence and double colour staining of platelets and leucocytes using FITC labeled anti-mouse CD41 (platelets) and PE labeled anti-mouse CD 45 (common leucocyte antigen) respectively. Our study has shown that the mean IPA for platelets stimulated by adenovirus was 2519.4 compared to 128.2 for resting platelets (n=5, p<0.02). Flow cytometric analysis of CD41 (platelets) and CD45 (leucocytes) double stained positive events indicated that Ad stimulation induced PLA when compared to the unstimulated samples. PLA visualisation was also confirmed by means of direct fluorescence microscopy. These preliminary in vitro results indicate that the Adenovirus activates mouse platelets as it induces degranulation and expression of P-selectin and supports the recruitment of leucocytes presumably through an interaction with the leucocyte P-selectin glycoprotein ligand-1. This may have a role in mediating the accelerated platelet clearance by the reticuloendothelial system following adenovirus administration. In addition, the interaction with leucocytes may also stimulate pro-coagulant microparticle generation and thus lead to systemic hemostatic activation. To our knowledge, this is the first report that identifies Adenovirus as an inducer for mouse platelet activation and as a promoter for platelet leucocyte association. However, further in vitro and in vivo studies are still needed to better understand the mechanism of Ad induced thrombocytopenia; a complication that significantly limits the application of Ad mediated gene therapy.

Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient mice.

Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE-/-TP-/- mice exhibited a significant delay in atherogenesis, and apoE-/-IP-/- mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE-/-IP-/- mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE-/-TP-/- mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE-/-IP-/- and apoE-/-TP-/- mice, respectively, than in those of apoE-/- mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE-/-IP-/- mice than in either apoE-/-TP-/- or apoE-/- mice. We conclude that TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Roles of thromboxane A2 and prostacyclin in the development of atherosclerosis in apoE-deficient mice

Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE–/–TP–/– mice exhibited a significant delay in atherogenesis, and apoE–/–IP–/– mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE–/–IP–/– mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE–/–TP–/– mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE–/–IP–/– and apoE–/–TP–/– mice, respectively, than in those of apoE–/– mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE–/–IP–/– mice than in either apoE–/–TP–/– or apoE–/– mice. We conclude that TXA2 promotes and PGI2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Control of platelet activation by cyclic AMP turnover and cyclic nucleotide phosphodiesterase type-3

Prostaglandin-induced cAMP elevation restrains key signaling pathways in platelet activation including Ca 2+ mobilization and integrin αIIbβ3 affinity regulation. We investigated how cAMP turnover by cyclic nucleotide phosphodiesterases (PDEs) regulates platelet activation. In washed human platelets, inhibition of all PDEs and also specific inhibition of PDE3 but not of PDE5 suppressed thrombin-induced Ca 2+ responses. The effect of general PDE or PDE3 inhibition was accompanied by an increase in cAMP, and potentiated by Gs stimulation with prostaglandin E 1. In platelet-rich plasma, general or PDE3 inhibition blocked platelet aggregation, integrin activation, secretion and thrombin generation. In contrast, inhibition of PDE5 increased the cGMP level, but without significant influence on aggregation, αIIbβ3 activation, secretion or procoagulant activity. Nitroprusside (nitric oxide) potentiated the effect of PDE5 inhibition in elevating cGMP. Nitroprusside inhibited platelet responses, but this was accompanied by elevation of cAMP. Together, these results indicate that cAMP is persistently formed in platelets, independently of agonist-induced Gs stimulation. PDE3 thus functions to keep cAMP at a low equilibrium level and reduce the cAMP-regulated threshold for platelet activation. This crucial role of PDE3, but not of PDE5, extends to all major processes in thrombus formation: assembly of platelets into aggregates, secretion of autocrine products, and procoagulant activity.

The study of serine-threonine kinases.

Serine-threonine kinases play crucial roles in activating and inhibitory signaling pathways that control platelet activation. Diversity in isozyme composition, catalytic and regulatory subunits, and subcellular localization provide a multitude of signaling properties through which the platelet initiates its functional responses or prevents them. This chapter describes a number of techniques and assays that may be used to study various specific serine-threonine kinases in platelets and their regulation during the platelet-activation process.

Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension

The binding of plasma von Willebrand factor (VWF) to platelet receptor GpIb under high hydrodynamic shear leads to platelet activation and subsequent shear-induced platelet aggregation (SIPA). We quantitatively examined the aspects of fluid flow that regulate platelet activation by subjecting human blood and isolated platelets to well-defined shear conditions in a cone-plate viscometer. We made the following observations. First, Annexin V binding to phosphatidyl serine expressed on activated cells was detectable within 10 seconds of shear application. Second, fluid shear stress rather than shear rate controls platelet activation, and a threshold shear stress of approximately 80 dyn/cm(2) is necessary to induce significant activation. Under these conditions, individual domains of soluble VWF and platelet GpIb are subjected to similar magnitudes of fluid forces on the order of 0.1 pN, whereas GpIb with bound VWF is subjected to 1 pN. Third, cell-cell collisions and time-varying stresses are not essential for platelet activation. Fourth, the mechanism of platelet activation can be resolved in 2 steps based on the contribution of VWF and fluid forces. Fluid shear and VWF are required during the first step, when GpIb-VWF binding likely occurs. Subsequently, high shear forces alone in the absence of VWF in suspension can induce platelet activation. In other experiments, purified VWF was subjected to shear in the viscometer, and VWF morphology was assessed using light scattering. These studies demonstrate, for the first time, the ability of hydrodynamic forces to induce VWF aggregation in suspension. This VWF self-association may be an additional feature involved in controlling cell adhesion rates in circulation.

C1 inhibitor infusion modifies platelet activity in hereditary angioedema patients.

C1 inhibitor (C1-INH) is an alpha2-globulin that blocks esterolytic activity of the first component of the classic complement cascade. The alpha-granules of normal human platelets also contain C1-INH, which is expressed on the platelet surface during platelet secretion in healthy patients, but it is clearly reduced in patients with hereditary angioedema (HAE). To evaluate the effects of in vivo C1-INH concentrate infusion on platelet responsiveness and coagulation system activity in patients with HAE. Assessment of the platelet activity and plasma levels of C1-INH, activated factor XII (XIIa), and prothrombin fragment F1.2 (F1.2) before and after infusion of 15 U/kg of C1-INH concentrate. In 6 patients (4 men and 2 women), HAE was diagnosed according to the accepted clinical and laboratory criteria. Platelet aggregation (final concentrations: adenosine diphosphate, 0.5, 1.25, and 2.5 microM; collagen, 5 microg/mL), C1-INH antigen (radial immunodiffusion), C1-INH activity (chromogenic substrates), and XIIa and F1.2 (enzyme-linked immunosorbent assay). After C1-INH infusion, we observed a prompt increase of C1-INH level and a slow return toward its plasma preinfusion values within 4 to 7 days, a significant decrease of both adenosine diphosphate- and collagen-induced platelet aggregation versus preinfusion values (maximum after 1-2 days; P <.001), and a rapid decrease of high basal values of XIIa and F1.2 in 30 and 120 minutes, respectively. These data show a role of C1-INH in the control of platelet activity and that its deficiency increases platelet aggregability and plasma levels of XIIa and F1.2 in patients with HAE.

Changes in the effects of interleukin-1β and tumor necrosis factor-α on platelet activation in early pregnancy

Platelets are known to be activated in normal pregnancy, and are further activated in pathological pregnancy states, such as preeclampsia. The factors controlling platelet activation are unknown, but cytokines, such as interleukin 1 β (IL-1 β ) and tumor necrosis- α (TNF- α ) have been found to affect platelet function and are believed to be involved in early pregnancy. We assessed the effects of these cytokines on platelets from women at various stages of pregnancy. We compared two methods: platelet in vitro aggregation by aggregometry, and platelet P-selectin expression by flow cytometry. IL-1 β and TNF- α had no effect on the in vitro aggregation and P-selectin expression of platelets from women in the first trimester of pregnancy as compared to the inhibitory effects of both in late pregnancy. We conclude that maternal platelet function undergoes a marked change throughout pregnancy.

In vivo formation of 8-iso-prostaglandin f2alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation.

Diabetes mellitus (DM) is associated with enhanced lipid peroxidation and persistent platelet activation. We tested the hypothesis that the in vivo formation of the F2-isoprostane 8-iso-prostaglandin (PG)F2alpha, a bioactive product of arachidonic acid peroxidation, is enhanced in DM and contributes to platelet activation. Urine samples were obtained from 85 diabetic patients and 85 age- and sex-matched healthy subjects for measurement of immunoreactive 8-iso-PGF2alpha and 11-dehydro-thromboxane B2 (TXM), an in vivo index of platelet activation. Sixty-two had non-insulin-dependent (NID)DM, and 23 had insulin-dependent (ID) DM. Vitamin E supplementation, metabolic control, and cyclooxygenase inhibitors were used to investigate the mechanisms of formation of 8-iso-PGF2alpha in this setting. Urinary 8-iso-PGF2alpha excretion was significantly higher (P=0.0001) in NIDDM patients (419+/-208 pg/mg creatinine; range 160 to 1014) than in age-matched control subjects (208+/-92; 41 to 433). Urinary 8-iso-PGF2alpha was linearly correlated with blood glucose and urinary TXM. 8-iso-PGF2alpha excretion was also significantly (P=0. 0001) higher in IDDM patients (400+/-146; 183 to 702) than in control subjects (197+/-69; 95 to 353). Vitamin E supplementation (600 mg/d for 14 days) was associated with a statistically significant reduction in both urinary 8-iso-PGF2alpha (by 37%) and TXM (by 43%) in 10 NIDDM patients. Improved metabolic control was associated with a significant (P=0.0001) reduction in 8-iso-PGF2alpha and TXM excretion by 32% and 41%, respectively, in 21 NIDDM patients. 8-iso-PGF2alpha was unchanged after 2-week dosing with aspirin and indobufen despite profound suppression of TXM excretion. We conclude that DM is associated with increased formation of F2-isoprostanes, as a correlate of impaired glycemic control and enhanced lipid peroxidation. This may provide an important biochemical link between impaired glycemic control and persistent platelet activation. These results provide a rationale for dose-finding studies of antioxidant treatment in diabetes.

Tyrosine Phosphorylation of the Novel Protein-tyrosine Kinase RAFTK during an Early Phase of Platelet Activation by an Integrin Glycoprotein IIb-IIIa-independent Mechanism

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.