Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation.

INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

Impact of atrial fibrillation on platelet gene expression.

AIMS: Platelets retain cytoplasmic messenger RNA and are capable of protein biosynthesis. Several diseases are known to impact the platelet transcriptome but the effect of non-valvular atrial fibrillation (NVAF) on platelet RNA transcript is essentially unknown. The aim of this study was to evaluate the impact of NVAF on platelet RNA transcript by measuring platelet genes expression in consecutive NVAF patients before and 3-4 months after pulmonary vein isolation (PVI) and compared to normal sinus rhythm controls (NSR). METHODS AND RESULTS: RNA from isolated platelets were reverse transcribed, assayed against 15 genes using real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. Expression of all evaluated genes, except cathepsin A gene, was significantly lower (higher ΔCt) in 103 NVAF patients compared to 55 NSR controls. Insulin-like growth factor binding protein acid labile subunit gene (IGFALS) had expression more than 16 fold-lower (17.0±2.8 vs 12.5±3.8, P<.001), follow by genes encoding for prostacyclin receptor, and for von Willebrand factor which had fourfold lower expression compared to NSR controls. Gender, type of atrial fibrillation, heart failure, hypertension, prior stroke, diabetes mellitus, and atherosclerosis were associated with different gene expression. Following PVI, expression of four genes significantly increased, particularly IGFALS gene (increased 256-fold) and ADAMT gene increased 16-fold); expression of three genes significantly decreased, and expression of eight genes has not changed. CONCLUSIONS: Platelets are capable to respond to the circulatory environment of NVAF by altering transcript and changing prothrombotic status. This shows platelet potential for molecular "reprogramming" possibly induced by flow disturbances of NVAF.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Platelet factor XIII gene expression and embolic propensity in atrial fibrillation.

Nearly 15% of patients with non-valvular atrial fibrillation (NVAF) have left atrial appendage thrombus (LAAT) by transesophageal echocardiography (TEE) and yet the annual stroke rate averages 5%. The aim of this study was to identify variables influencing embolic propensity of LAAT. Platelet RNA was extracted from platelet-rich regions within formalin-fixed, paraffin-embedded specimens obtained from NVAF patients during cardiac surgery (26 LAAT from 23 patients) or peripheral embolectomy (51 thrombi from 41 patients). Platelet RNA was also assessed from whole blood from 40 NVAF patients. Expression of six platelet-predominate genes: H2A histone family, A1 domain of factor XIII, integrin α2bß3; glycoprotein IX, platelet factor 4, glycoprotein Ib, was performed using TaqMan MGB-probe based quantitative real-time polymerase chain reaction. Platelet factor XIII subunit A gene expression was significantly lower in embolised compared to non-embolised thrombi as determined by normalised cycle threshold values (4.0 ± 1.2 v 2.8 ± 1.8, p=0.02). Expression of other genes did not differ by embolic status. In conclusion, RNA extracted from formalin-fixed, paraffin-embedded platelet-rich tissues can be used for analysis of platelet-predominate gene expression. Variable factor XIII gene expression in thrombi generated during NVAF may in part explain the propensity to embolisation.

Direct thrombin inhibitors are not equally effective in vivo against arterial thrombosis: in vivo evaluation of DuP714 and argatroban in a porcine angioplasty model and comparison to r-hirudin.

BACKGROUND: Qualitative differences in antithrombotic efficacy between thrombin inhibitors may be explained by the affinity for which they bind thrombin. This affinity is inversely proportional to the inhibitory constant for the agent (Ki). Thrombin inhibitors, DuP714 (Ki=10(-11)) and argatroban (Ki=10(-8)), were compared to our previous studies with r-hirudin (Ki=10(-13)). METHODS AND RESULTS: Prior to balloon angioplasty, thirty pigs randomly received DuP714 (0.1 mg/kg bolus and 0.6 mg/kg/h infusion; n=8), argatroban (0.2 mg/kg/min. continuous infusion; n=9), or saline (n=17). Injured arterial segments were measured for (111)In-platelet and 125I-fibrin(ogen) deposition and the incidence of macroscopic thrombus. In DuP714-treated animals, platelet and fibrin(ogen) deposition were significantly lower than controls in both carotid (10+/-2 vs. 62+/-18 and 20+/-4 vs. 74+/-6) and coronary (10+/-4 vs. 160+/-63 and 17+/-3 vs. 86+/-22) arteries (p<0.005). In contrast, platelet and fibrin(ogen) deposition were similar when comparing argatroban to saline in carotid (41+/-20 vs. 40+/-9 and 71+/-5 vs. 49+/-7) and coronary (92+/-33 vs. 151+/-45 and 114+/-37 vs. 89+/-38) arteries (p=0.82 and 0.38, respectively). Compared to argatroban, fibrin(ogen) (p<0.001) and coronary platelet deposition (p<0.05) were significantly reduced in animals treated with DuP714 with no significant difference in carotid platelet deposition (p=0.10). Neither inhibitor prevented macroscopic thrombosis. In previous studies with r-hirudin in this model, platelet deposition was limited to a monolayer with complete inhibition of macroscopic thrombus. CONCLUSIONS: Direct thrombin inhibitors do not equally prevent arterial thrombosis. Qualitative differences may be explained in part by the affinity for which they bind thrombin.