Platelet Recruitment Research Articles

Platelet recruitment to venous stent thrombi

Thrombosis following venous stent placement is a morbid clinical outcome. Whether to target platelets or coagulation factors for venous stent thromboprophylaxis remains unclear. We sought to determine whether integrin α(IIb)β3 antagonism with lamifiban would inhibit platelet recruitment to venous stent thrombosis. Anti-thrombotic efficacy was compared between venous and arterial circulations. Pigs received either lamifiban (0.2 mg/kg bolus plus 0.2 mg/kg/h infusion; n = 6) or saline (n = 12). Carotid arteries were crush injured and then harvested 30 min later to provide an assessment of antithrombotic efficacy in the arterial circulation. Iliac venous stents were then deployed and thrombi allowed to propagate for 2 h before harvesting. Platelet deposition was measured by scintillation detection of autologous (111)In-platelets. Venous thrombi were quantified by weight and compared to platelet, Von Willebrand factor (VWF) and fibrinogen content. Arterial platelet deposition (×10(6)/cm(2)) was reduced >80% by lamifiban (398 ± 437) compared to controls (1,540 ± 883; p < 0.005). Lamifiban also reduced venous thrombus platelet deposition (139 ± 88 vs. 281 ± 167) however did not prevent thrombosis. In control animals, venous stent platelet deposition correlated with plasma fibrinogen content (R(2) = 0.29; p = 0.03). Fibrinogen content correlated directly with venous stent platelet deposition (p = 0.03) but not thrombus weight. Neither venous platelet deposition nor thrombus weights varied by VWF content. Platelet recruitment to venous stent thrombi occurs in part through the integrin α(IIb)β3 receptor. Unlike arterial thrombosis, inhibition of this receptor is insufficient to prevent venous stent thrombosis.

Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (106/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s−1). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

Neutrophils Recruited to Sites of Infection Protect from Virus Challenge by Releasing Neutrophil Extracellular Traps

Neutrophils mediate bacterial clearance through various mechanisms, including the release of mesh-like DNA structures or neutrophil extracellular traps (NETs) that capture bacteria. Although neutrophils are also recruited to sites of viral infection, their role in antiviral innate immunity is less clear. We show that systemic administration of virus analogs or poxvirus infection induces neutrophil recruitment to the liver microvasculature and the release of NETs that protect host cells from virus infection. After systemic intravenous poxvirus challenge, mice exhibit thrombocytopenia and the recruitment of both neutrophils and platelets to the liver vasculature. Circulating platelets interact with, roll along, and adhere to the surface of adherent neutrophils, forming large, dynamic aggregates. These interactions facilitate the release of NETs within the liver vasculature that are able to protect host cells from poxvirus infection. These findings highlight the role of NETs and early tissue-wide responses in preventing viral infection.

Mild Hypercholesterolemia Blunts the Proinflammatory and Prothrombotic Effects of Hypertension on the Cerebral Microcirculation

Although an increased leukocyte and platelet adhesion is observed in cerebral venules of mice with either hypertension (HTN) or hypercholesterolemia (HCh), it remains unclear whether the combination of HTN and HCh exerts a comparable effect on leukocyte and platelet recruitment in the cerebral microvasculature. Thus, we examined whether HCh alters platelet and leukocyte adhesion, and blood-brain barrier (BBB) permeability, in cerebral venules in two models of murine HTN: DOCA salt-induced and angiotensin II (Ang II) induced. In both models, the mice were placed on either a normal or cholesterol-enriched diet. An enhanced recruitment of adherent leukocytes and platelets in cerebral venules was noted in both HTN models in the absence of HCh, but not in its presence. The Ang II-induced increase in BBB permeability was attenuated by HCh as well. Both total and high-density lipoprotein (HDL) cholesterol levels were elevated in the HCh mice. The HTN-induced increase in leukocyte and platelet adhesion was attenuated in apolipoprotein A-I transgenic mice (ApoA1-Tg) and blunted in wild-type mice treated with the ApoA1 mimetic peptide, 4F. Our findings indicate that mild HCh significantly blunts the cerebral microvascular responses to HTN and that HDL may have a role in mediating this beneficial effect of HCh.

Platelet Reactivity and CD39 Ectonucleotidase Activities in Cryptogenic Stroke

Abstract 3397 Background:In the US, 795,000 patients are diagnosed with stroke annually. Of the 90% of strokes that are ischemic, a substantial number are of unknown etiology. In younger adults, the proportion of such cryptogenic strokes exceeds 40%. Cryptogenic stroke patients have an increased prevalence of interatrial shunts (IAS), which can be found in as many as 60% of cases as compared to 26% for the general population. Yet, percutaneous closure of such defects has not been found to reduce the recurrence rate as compared with medical therapy. Hypercoagulable disorders have also been implicated in the pathogenesis of premature ischemic strokes, but their occurrence in cryptogenic stroke series is modest, leaving many such strokes unexplained. We hypothesized that cryptogenic stroke may in part represent a disorder of enhanced platelet reactivity. An understanding of the responsiveness of platelets from young cryptogenic stroke patients to a broad panel of agonists is required because platelet reactivity to a single agonist or a thromboxane measurement does not provide sufficient information about platelet reactivity. CD39/NTPDase1, the ecto-enzyme on endothelial cells and leukocytes, is responsible for control of adenine nucleotide levels in the local vascular microenvironment, and, therefore, blood fluidity. CD39 rapidly metabolizes ATP and ADP released from activated platelets and other cells, thereby abolishing the prothrombotic cascade of platelet activation and recruitment. Methods:We sought to determine whether platelet activation and recruitment, markers of platelet activation, and CD39 enzymatic activity are increased in younger patients with unexplained stroke. To this end, we evaluated such platelet-related measures in a subsample (n=167 subjects) participating in the THrombophilia In Cryptogenic stroKe (THICK) study, a prospective case-control study evaluating the prothrombotic determinants of unexplained stroke. Cases consisted of patients, ages 18–64, referred to Weill Cornell Medical Center for evaluation of cryptogenic stroke, whereas controls were stroke-free volunteers within the same age range recruited at our center and the surrounding area. Cryptogenic stroke was defined according to modified TOAST criteria. Results:Cases (n=99) were similar in age (median 45 years old), sex (51% women), and race-ethnic composition (8% African American) as compared with controls (n=68). They also had similar prevalences of hypertension (18%), diabetes (7%), dyslipidemia (40%), and current smoking (6%). Cases had over twice the prevalence of IAS (56%) as controls (24%). Extensive testing for humoral prothrombotic disorders revealed a potential explanation for stroke in only half of the patients. We developed a Broad Range Aggregometry System to study profiles of platelet reactivity to multiple agonists, and classified patient responsiveness based on their reactivity to these agonists. We have also been able to make a laboratory diagnosis of aspirin non-responsiveness in these patients and controls. In addition, in stroke subjects, trends to higher total ADPase activity were observed on lymphocytes (p=0.09) and PMN (p=0.09), while ATPase activities were similar (p=0.81 and 0.68, respectively), as determined by our radio-TLC nucleotidase assay. Also, the ADPase to ATPase activity ratio was greater in the stroke patients than controls (p=0.003 for lymphocytes; p=0.13 for PMN). Cryptogenic stroke patients also displayed increased levels of platelet-monocyte aggregates (p=0.034) and the platelet activation marker CD63 (p=0.048), but not CD154 (p=0.183), as compared to controls (determined by FACS analyses). Conclusions:These findings support a potential role for enhanced platelet reactivity and a low threshold for platelet responsiveness in cryptogenic stroke patients. Disclosures:No relevant conflicts of interest to declare.

Platelet α-Granule Secretion and Cytoskeletal Rearrangements Are Spatially Regulated At the Micro/Nanoscale.

Abstract 2163 IntroductionAt sites of vascular injury, activated platelets exhibit dramatic morphological changes and granule secretion to facilitate recruitment of other platelets and clot formation. In our previous work (Kita et al., 2011), we quantified the effect of the microenvironmental geometry on platelet adhesion using microcontact printing and showed that platelet adhesion and spreading is spatially regulated with microscale resolution. Here we demonstrate that platelet secretion of alpha granules, as indicated with P-selectin staining (a marker for a-granules), is also spatially regulated at the micro/nanoscale. Specifically, we show that fibrinogen micropatterns regulate and determine the spatial distribution of P-selectin at the single platelet level. We also show that tubulin, one of the major components of the platelet cytoskeleton, is also rearranged by the nanoscale geometry of the microenvironment. MethodsUsing microfabrication techniques we previously developed (Kita et al., 2011), patterned polydimethylsiloxane (PDMS) stamps were “inked” with fluorescently-labeled fibrinogen from human plasma. Fibrinogen patterns were then “microstamped” onto glass coverslips and the printed surface was blocked with 1 % BSA. 20 million/ml of washed human platelets were prepared in Tyrode’s buffer and incubated onto protein micropatterned surfaces. Immunofluorescence staining was used to visualize P-selectin surface expression and intracellular distribution, and tubulin distribution of adhered platelets. Platelets were also counter-stained with a fluorescent membrane dye or phalloidin for cell detection or cytoskeleton arrangement, respectively. Images were taken via confocal microscopy with a 63x oil immersion objective. ResultsWhen washed platelets are incubated onto the micropatterned fibrinogen surface, they generally undergo activation and lamellipodia formation. On uniform, non-patterned fibrinogen glass surfaces, (Fig 1A), the average area of spread platelets is 24.86 μm2(n=30, standard error ± 2.27). When the fibrinogen micropattern was larger than the platelets’ average area (Fig 1B), multiple platelets covered the micropattern and followed the microenvironmental geometry with high fidelity. P-selectin was detected on the entire platelet surface at slightly higher concentrations at the margins. However, when the features of the fibrinogen micropattern decreased to 5 μm in diameter (Fig 1C, D, and Fig2), platelet spreading was not constrained within the micropattern boundaries. Interestingly, the area of the platelets that spread beyond the fibrinogen micropattern exhibited much higher P-selectin expression than the areas atop the fibrinogen micropattern (Fig 2, arrows), indicating that expression and distribution of P-selectin are spatially regulated by the geometry of the fibrinogen substrate at the microscale.In addition, when platelets are incubated onto fibrinogen microstamps with densely spaced “holes” (0.4–1.0 μm in diameter) blocked with BSA, platelets fully spread and span over those holes. However, dense P-selectin expression was co-localized with these holes, indicating that P-selectin expression and a-granule release are spatially regulated by the underlying protein micropattern (Fig 3A). Interestingly, tubulin showed the opposite trend and only localized to areas directly above the fibrinogen micropattern (Fig 3B). These observations suggest that both platelet a-granule distribution/secretion and cytoskeleton arrangement are regulated at the single platelet level with nanoscale resolution (Fig 3C). ConclusionsUsing our platelet adhesion/microstamping technique, we demonstrated that platelets regulate the intracellular trafficking, distribution, and secretion of biomolecules at the nanoscale, responding to the geometry of microenvironment. We will continue to investigate how platelets spatially regulate other aspects of their physiology, including calcium signaling and distribution of receptors/ligands on different substrates such as vWF and collagen. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Leukocyte-Dependent Responses of the Microvasculature to Chronic Angiotensin II Exposure

Angiotensin II (Ang II) contributes to the pathogenesis of hypertension and other cardiovascular diseases. Ang II induces a pro-oxidative, proinflammatory, and prothrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, it remains unclear whether and how the blood cell recruitment is linked to the production of reactive oxygen species. In this study, we addressed the contributions of Ang II type 1 receptors (AT(1)r) and gp91(phox) to the recruitment of leukocytes and platelets and reactive oxygen species production in venules during chronic (2-week) infusion of Ang II in wild-type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle postcapillary venules. In WT mice, Ang II infusion induced a time-dependent increase in the adhesion of leukocytes and platelets and enhanced reactive oxygen species production in venules. These changes in blood cell adhesion and reactive oxygen species production were not observed in AT(1)r(-/-) mice, AT(1)r(-/-) bone marrow chimeras (blood cells deficient in AT(1)r), gp91(phox-/-) mice, gp91(phox-/-) chimeras (blood cells or endothelial cells deficient in gp91(phox)), and in WT mice rendered granulocytopenic via intraperitoneal injection of antimouse granulocyte receptor 1 antibody. Thrombocytopenic WT mice (platelets depleted by intraperitoneal injection of rabbit antimouse thrombocyte antiserum) responded similar to WT mice. These findings implicate leukocyte-associated AT(1)r and gp91(phox) in the induction of the pro-oxidative, proinflammatory, and prothrombogenic phenotype assumed by microvessels that is chronically exposed to elevated Ang II.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

C0195 The presence of the TXA2 receptor in lipid rafts of platelets is necessary for platelet activation by TXA2

Thromboxane A2 (TXA2) synthesized by activated platelets plays an important role in the pathophysiology of thrombogenesis, by acting on its specific receptor (TXA2-R). This reinforces the activation of platelets that produce it and promotes platelet recruitment and thrombus growth. Lipid rafts are cholesterol-rich microdomains present in the plasma membrane which concentrate signaling molecules and receptors, and play an important role in signal transduction in platelets. Currently it has not been described the presence of TXA2-R associated with lipid rafts in human platelets.

Rosuvastatin reduces platelet recruitment by inhibiting NADPH oxidase activation

Rosuvastatin increased vascular endothelial NO and attenuated platelet activation after ischemia-reperfusion in mice; nevertheless, the influence of rosuvastatin on the activation of human platelets and the underlying mechanism has never been investigated.In an in vitro study platelets from 8 healthy donors were incubated with scalar concentrations of rosuvastatin (1–10μM) before activation. Platelet recruitment (PR), that mimics the propagation of platelet aggregation and is dependent upon isoprostane formation, was investigated. PR was inhibited by rosuvastatin in concentration-dependent manner concomitantly with down-regulation of platelet release of the pro-thrombotic molecule CD40L. This effect was associated with lower production of platelet reactive oxygen species (ROS), isoprostane and activation of the glycoprotein IIb/IIIa and was counteracted by exogenous addition of isoprostanes. Conversely, rosuvastatin concentration-dependently increased platelet NO. Platelet isoprostane formation mainly depends from NADPH oxidase. Rosuvastatin concentration-dependently inhibited platelet sNOX2-dp release, a specific marker of NADPH oxidase activation, PKC phosphorylation and p47phox translocation from cytosol to membranes.In an ex vivo study 10 hypercolesterolemic patients were randomly allocated to diet or rosuvastatin (20mg). We observed that as early as 2h after rosuvastatin PR, platelet isoprostanes formation, platelet CD40L and sNOX2-dp decreased while platelet NO increased; no changes were detected in diet-assigned patients.This study shows that in vitro rosuvastatin impairs platelet activation via inhibition of NOX2-derived oxidative stress. This effect, which is associated ex vivo with acute inhibition of platelet activation, suggests that rosuvastatin behaves as an antiplatelet drug.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.

A brief review of the past and future of platelet P2Y12 antagonist

ADP plays a pivotal role in localized platelet activation and recruitment, and, with that, in the maintenance of thrombus integrity, making it a suitable target for the control of intravascular thrombosis. The limited distribution of one of its receptors, the P2Y12 receptor, primarily to platelets makes it an especially attractive pharmacologic target. For the last several decades the thienopyridine family of P2Y12 antagonists have provided the vast majority of clinical data confirming the clinical benefit of selective P2Y12 inhibition. Recently, new thienopyridine plus nonthienopyridine P2Y12 antagonists have become available or are being studied that will further improve our treatment of patients with coronary disease.

Immediate Antioxidant and Antiplatelet Effect of Atorvastatin via Inhibition of Nox2

Statins exert an antithrombotic effect in patients at risk of or with acute thrombosis, but no study has investigated whether this effect is immediate and whether there is an underline mechanism. Patients with hypercholesterolemia were randomly allocated to a Mediterranean diet with low cholesterol intake (<300 mg/d; n=15) or atorvastatin (40 mg/d; n=15). Oxidative stress, as assessed by serum Nox2 and urinary isoprostanes, and platelet activation, as assessed by platelet recruitment, platelet isoprostanes, and thromboxane A(2), platelet Nox2, Rac1, p47(phox), protein kinase C, vasodilator-stimulated phosphoprotein, nitric oxide, and phospholipase A(2), were determined at baseline and after 2, 24, and 72 hours and 7 days of follow-up. An in vitro study was also performed to see whether atorvastatin affects platelet oxidative stress and activation. The atorvastatin-assigned group showed a significant and progressive reduction of urinary isoprostanes and serum Nox2, along with inhibition of platelet recruitment, platelet isoprostanes, Nox2, Rac1, p47(phox), and protein kinase C, starting 2 hours after administration. Platelet phospholipase A(2) and thromboxane A(2) significantly decreased and vasodilator-stimulated phosphoprotein and nitric oxide increased after 24 hours. Low-density lipoprotein cholesterol decreased significantly after 72 hours and further declined after 7 days. No changes were observed in the Mediterranean diet group. In vitro experiments demonstrated that atorvastatin dose-dependently inhibited platelet Nox2 and phospholipase A(2) activation, along with inhibition of platelet recruitment, platelet isoprostanes, and thromboxane A(2), and increased vasodilator-stimulated phosphoprotein and nitric oxide. The study provides the first evidence that atorvastatin acutely and simultaneously decreases oxidative stress and platelet activation by directly inhibiting platelet Nox2 and ultimately platelet isoprostanes and thromboxane A(2). These findings provide a rationale for the use of statins to prevent or modulate coronary thrombosis. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01322711.

Donor safety in triple plateletpheresis: results from the German and Austrian Plateletpheresis Study Group multicenter trial

The objective was to investigate potential risks for apheresis donors associated with a triple-plateletpheresis (TP) program. Eleven hemapheresis centers randomly assigned 411 repeat donors (ratio, 1:1.2) to either double plateletpheresis (DP; 185 donors) or TP (226 donors) with a platelet (PLT) target content of at least 5.0×10(11) PLTs/DP and at least 7.5×10(11) PLTs/TP. The primary endpoint was procedure-related postapheresis PLT count of at least 150×10(9) /L (probability, ≥98%). Secondary endpoints were apheresis characteristics and donor adverse reactions. In 6 of 1133 DPs (0.5%) in 4 of 185 donors (2.2%) and in 20 of 1020 TPs (2.0%) in 14 of 226 donors (6.2%), postapheresis PLT counts were below 150×10(9) /L. There were marginal but significant differences in collection efficiency (DP, 69.2±9.1%; TP, 70.9±9.0%; p≤0.0001) and collection rate (DP, 10.4×10(9) ±2.3×10(9) PLTs/min; TP, 10.8×10(9) ±2.3×10(9) PLTs/min; p≤0.005). The PLT yields were 5.9×10(11) ±0.8×10(11) PLTs for DP and 8.3×10(11) ±0.9×10(11) PLT for TP (p≤0.0001) at processing times of 59±13 minutes (DP) versus 80±16 minutes (TP; p≤0.0001). Significant PLT recruitment (1.10±0.14 vs. 1.20±0.23; p<0.0001) was seen for both DP and TP. DP and TP did not differ with regard to venous access problems (VAPs) without discontinuation (3.8% for both), but DP induced fewer VAPs with discontinuation (1.1% vs. 3.0%; p<0.01). Mild citrate toxicity (1.7% vs. 3.9%; p<0.01) and circulatory reactions (0.4% vs. 2.2%; p<0.01) were more often noticed in TP, but caused no increase in discontinuations. TP results in an increase in mild donor reactions but does not significantly impair donor safety or product quality.

Ectonucleotide Triphosphate Diphosphohydrolase-1 (CD39) Mediates Resistance to Occlusive Arterial Thrombus Formation after Vascular Injury in Mice

Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/β(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.

Inherited platelet disorders and oral health

Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention.

Platelet Isoprostane Overproduction in Diabetic Patients Treated With Aspirin

Aspirin modestly influences cardiovascular events in patients with type 2 diabetes mellitus (T2DM), but the reason is unclear. The aim of the study was to determine whether in T2DM patients aspirin enhances platelet isoprostanes, which are eicosanoids with proaggregating properties derived from arachidonic acid oxidation by platelet NOX2, the catalytic subunit of reduced NAD phosphate oxidase. A cross-sectional study was performed comparing T2DM patients, treated (n = 50) or not treated (n = 50) with 100 mg/day aspirin, with 100 nondiabetic patients, matched for age, sex, atherosclerosis risk factors, and aspirin treatment. A short-term (7 days) treatment with 100 mg/day aspirin also was performed in 36 aspirin-free diabetic and nondiabetic patients. Higher platelet recruitment, platelet isoprostane, and NOX2 activation was found in diabetic versus nondiabetic patients and in aspirin-treated diabetic patients versus nontreated patients (P < 0.001). Platelet thromboxane (Tx) A2 (P < 0.001) was inhibited in all aspirin-treated patients. In the interventional study, aspirin similarly inhibited platelet TxA2 in diabetic and nondiabetic patients (P < 0.001). Platelet recruitment, isoprostane levels, and NOX2 activation showed a parallel increase in diabetic patients (P < 0.001) and no changes in nondiabetic patients. These findings suggest that in aspirin-treated diabetic patients, oxidative stress–mediated platelet isoprostane overproduction is associated with enhanced platelet recruitment, an effect that mitigates aspirin-mediated TxA2 inhibition.

Identification of Different Proaggregatory Abilities of Activated Platelet Subpopulations

Blood platelets are anucleate cell fragments that play a critically important role in hemostasis and thrombosis. Platelets are activated with various agonists that allow them to aggregate, thus forming either hemostatic plugs or pathologic thrombi. Recent studies have revealed that at least two activated platelet subpopulations are formed upon potent stimulation of platelets with collagen and/or thrombin. One of these subpopulations consists of so-called coated platelets that express high levels of phosphatidylserine and retain α-granule proteins, including fibrinogen, on their surface. They also have reduced levels of the main aggregation receptor-activated glycoprotein IIb-IIIa, which might indicate a defect in their proaggregatory ability. In this study, the proaggregatory abilities of coated and noncoated platelets were assessed by means of light transmission aggregometry of suspensions with varying ratios of platelets from one subpopulation to those of a different subpopulation. A mathematical model of platelet aggregation in heterogeneous mixtures was developed to assist in the analysis of experimental data. Flow cytometry was employed to monitor platelet recruitment into aggregates and the ability of platelets to bind external fibrinogen. Finally, confocal microscopy was used to image coated platelets involved into aggregates formed by mechanical shaking. The obtained data revealed to our knowledge a novel mechanism regulating aggregate formation of platelet subpopulations: coated platelets cannot aggregate with each other but can be recruited into aggregates by noncoated platelets.

Abstract 220: Interleukin 17 Promotes Endothelial Activation and Neutrophil Recruitment Through Synergistic Action with Tumor Necrosis Factor α

Introduction: Interleukin 17 (IL-17) is the signature cytokine produced by TH17 cells, a novel subset of pro-inflammatory CD4+ T cells. Prior studies have noted elevated serum IL-17 in patients presenting with acute coronary syndrome but little is known regarding the effect of IL-17 on endothelial (EC) activation, a key step in thrombosis and the process of leukocyte and platelet recruitment to sites of inflammation. Methods and results: We tested the hypothesis that IL-17 would induce a distinct pattern of EC activation and leukocyte recruitment when compared to the TH1 cytokine IFNγ. We found that activation of mouse heart EC with the combination of TNFα and IL-17 but not IFNγ had a synergistic effect on mRNA expression of P-selectin (2.6 fold over TNFα alone, p&lt;0.01) and E-selectin (4.0 fold, p&lt;0.01); as well as the CXCR2-ligands CXCL1 (29.0 fold, p&lt;0.01), CXCL2 (15.2 fold, p&lt;0.01), and CXCL5 (53.4 fold, p&lt;0.05), which are highly chemotactic for neutrophils (PMN). Furthermore, EC activation with TNFα and IL-17 but not IFNγ had a synergistic effect on the level of PMN transmigration (46.9% versus 9.1% for TNFα alone, p&lt;0.01) in a parallel-plate flow chamber model, which was prevented upon perfusion of PMN from CXCR2 (-/-) mice. In addition, TNFα and IL-17 had a synergistic effect on microvascular inflammation in vivo as determined by intravital microscopy of cremasteric leukocyte rolling velocity (8.9 μm/s versus 23.9 μm/s for TNFα alone, p&lt;0.01) and perivascular infiltration (22.4 cells/field versus 13.9 cells/field, p&lt;0.01) at 16hr after cytokine injection. By contrast, treatment with TNFα and IFNγ but not IL-17 induced the expression of ICAM1 (2.0 fold over TNFα alone, p&lt;0.05) and VCAM1 (1.6 fold, p&lt;0.01), as well as the T-cell chemokines, CXCL9 (187.4 fold, p&lt;0.01), CXCL10 (2.9 fold, p&lt;0.01), and CCL5 (2.6 fold, p&lt;0.01). Accordingly, treatment with TNFα and IFNγ but not IL-17 preferentially enhanced the transmigration of effector CD4+ T cells under shear flow (41.9% versus 29% for TNFα alone, p&lt;0.01). Conclusion: IL-17 and TNFα act in a synergistic manner to induce a pattern of EC activation that favors selective PMN recruitment to sites of inflammation, providing a potential link between IL-17 and risk of atherosclerotic plaque rupture and thrombosis.