Chris I. Jones

Beneficial effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy.

Background—Postoperative thromboembolic stroke affects 2% to 3% of patients undergoing carotid endarterectomy (CEA) and is preceded by 1 to 2 hours of increasing cerebral embolization. Previous work has demonstrated that high rates of postoperative embolization are associated with increased platelet reactivity to adenosine 5′-diphosphate (ADP). Our hypothesis was that preoperative administration of the platelet ADP antagonist clopidogrel could reduce postoperative embolization. Methods and Results—One hundred CEA patients on routine aspirin therapy (150 mg) were randomized to 75 mg clopidogrel (n=46) or placebo (n=54) the night before surgery. Platelet response to ADP was assessed by whole-blood flow cytometry. The number of emboli detected by transcranial Doppler within 3 hours of CEA was independently quantified. Time taken from flow restoration to skin closure was used as an indirect measure of the time to secure hemostasis. In comparison with placebo, clopidogrel produced a small (8.8%) but significant reduction in the platelet response to ADP (P <0.05) while conferring a 10-fold reduction in the relative risk of those patients having >20 emboli in the postoperative period (odds ratio, 10.23; 95% CI, 1.3 to 83.3; P =0.01, Fisher’s exact test). However, in the clopidogrel-treated patients, the time from flow restoration to skin closure (an indirect marker of hemostasis) was significantly increased (P =0.04, Fisher’s exact test), although there was no increase in bleeding complications or blood transfusions. Conclusions—This is the first study to show that a CEA patient’s postoperative thromboembolic potential can be significantly reduced by targeted preoperative antiplatelet therapy without increasing the risk of bleeding complications.

A novel variant on chromosome 7q22.3 associated with mean platelet volume, counts, and function.

Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 +/- 0.001 log fL; P < 1.08 x 10(-24)) and PLT (per-G effect -4.55 +/- 0.80 10(9)/L; P < 7.19 x 10(-8)) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis.

Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation

The thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re‐associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell‐surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet‐derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet‐surface thiol isomerases in the regulation of haemostasis.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus

Summary.  Background: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. Objective: To develop a methodology suitable for measuring signaling pathway‐specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. Methods: Three established platelet assays were evaluated: mobilization of [Ca2+]i, aggregometry and flow cytometry, each in response to adenosine 5′‐diphosphate (ADP) or the glycoprotein (GP) VI‐specific crosslinked collagen‐related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P‐selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter‐individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. Results: Individuals were identified who were hypo‐ or hyper‐responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r2 = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for ∼35% of the variation in the CRP‐XL response. Conclusion: Genotyping‐phenotype association studies in a well‐characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

Gap Junctions and Connexin Hemichannels Underpin Hemostasis and Thrombosis

Background— Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. Methods and Results— We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. Conclusions— Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

LXR as a novel antithrombotic target

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-β is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease.

Non-genomic effects of PPARgamma ligands: inhibition of GPVI-stimulated platelet activation.

Summary.  Background: Peroxisome proliferator‐activated receptor‐γ (PPARγ) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. Objective: In the present study, we aimed to demonstrate the ability of PPARγ ligands to modulate collagen‐stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. Methods: Washed platelets were stimulated with PPARγ ligands in the presence and absence of PPARγ antagonist GW9662 and collagen‐induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura‐2AM‐loaded platelets and tyrosine phosphorylation levels of receptor‐proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPARγ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. Results: PPARγ ligands inhibited collagen‐stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P‐selectin exposure. PPARγ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPARγ agonists, implicating PPARγ in the effects observed. Furthermore, PPARγ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPARγ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPARγ agonists, indicating a potential mechanism for PPARγ function in collagen‐stimulated platelet activation. Conclusions: PPARγ agonists inhibit the activation of collagen‐stimulation of platelet function through modulation of early GPVI signalling.

Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?

The majority of patients who suffer peri-operative thromboembolic complication while undergoing vascular procedures do so despite taking aspirin. This study examined the antiplatelet effect of aspirin during surgery in patients undergoing carotid endarterectomy (CEA). Fifty patients undergoing CEA were standardised to 150 mg aspirin daily for > or =2 weeks. Platelet aggregation in response to arachidonic acid (AA) was measured in platelet rich plasma prepared from blood taken prior to, during, and at the end of surgery. Spontaneous platelet aggregation was also studied, as was the role of physiological agonists (ADP, collagen, thrombin, and epinephrine) in mediating the in vivo and in vitro responses to AA. Eighteen patients undergoing leg angioplasty, also on 150 mg aspirin, without general anaesthesia, served as a control group. In the CEA patients aggregation induced by AA (5 mM) increased significantly from 7.6 +/- 5.5% pre-surgery to 50.8 +/- 29.5% at the end of surgery (p <0.0001). Aggregation to AA was even greater in samples taken mid-surgery from a sub-set of patients (73.8+/-7.2%; p = 0.0001), but fell to 45.9 +/- 7.4% by the end of surgery. The increased aggregation in response to AA was not due to intra-operative release of physiological platelet agonists since addition of agents that block/neutralise the effects of ADP (apyrase; 4 micro g/ml), thrombin (hirudin; 10 units/ml), or epinephrine (yohimbine; 10 micro M/l) to the samples taken at the end of surgery did not block the increased aggregation. The patients undergoing angioplasty also showed a significant rise in the response to AA (5 mM), from 5.6 +/- 5.5% pre-angioplasty to 32.4 +/- 24.9% at the end of the procedure (p <0.0001), which fell significantly to 11.0 +/- 8.1% 4 hours later. The antiplatelet activity of aspirin, mediated by blockade of platelet arachidonic acid metabolism, diminished significantly during surgery, but was partially restored by the end of the procedure without additional aspirin treatment. This rapidly inducible and transient effect may explain why some patients undergoing cardiovascular surgery remain at risk of peri-operative stroke and myocardial infarction.