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International audience; Aim: We report the study of a familial rare disease with recurrent venous thromboembolic events that remained undiagnosed for many years using standard coagulation and hemostasis techniques. Methods: Exome sequencing was performed in three familial cases with venous thromboembolic disease and one familial control using NimbleGen exome array. Clot lysis experiments were performed to analyze the reasons of the altered fibrinolytic activity caused by the mutation found. Results: We found a mutation that consists of a R458C substitution on the fibrinogen alpha chain (FGA) gene confirmed in 13 new familial subjects that causes a rare subtype of dysfibrinogenemia characterized by venous thromboembolic events. The mutation was already reported to be associated with a fibrinogen variant called fibrinogen Bordeaux. Clot-lysis experiments showed a decreased and slower fibrinolytic activity in carriers of this mutation as compared to normal subjects, thus demonstrating an impaired fibrinolysis of fibrinogen Bordeaux. Conclusions: The exome sequencing and clot-lysis experiments might be powerful tools to diagnose idiopathic thrombophilias after an unsuccessful set of biochemical laboratory tests. Fibrinogen Bor-deaux is associated with impaired fibrinolysis in this family with idiopathic thrombophilia.

Background and Purpose— There is a great interindividual variability among patients with acute ischemic stroke regarding the response to intravenous tissue-type plasminogen activator treatment. The aim of this study was to identify genetic variants associated with recanalization, and thus treatment efficacy, after tissue-type plasminogen activator administration. Methods— A total of 140 single nucleotide polymorphisms from 97 candidate genes were successfully genotyped by SNPlex in 2 cohorts, accounting for 497 prospectively recruited tissue-type plasminogen activator-treated patients, of whom 33% recanalized during tissue-type plasminogen activator infusion. Functional studies were then performed, including assessment of interleukin 1B mRNA levels and von Willebrand factor, FIII, FVII, FVIII, and FX protein activity. Results— After replication, the following single nucleotide polymorphisms were associated with early recanalization: rs1143627 in IL1B gene (CC: 53.1% of recanalization, A-carriers: 32.7%; P =0.022; replication cohort: P =0.046), rs16944 in IL1B gene (AA: 50% of recanalization, G-carriers: 32%; P =0.038; replication cohort: P =0.049), and rs1063856 in the vWF gene (GG: 53.8% of recanalization, A-carriers: 31.5%; P =0.006; replication cohort: P =0.046). The functional studies revealed an association between the rs1063856 single nucleotide polymorphisms in vWF and FVIII activity (AA: 115.93%, AG: 156.07%, GG: 83.42%; P =0.005). Conclusions— Three single nucleotide polymorphisms were associated with tissue-type plasminogen activator efficacy in the Spanish population, and their mechanism of action might be associated with the activity of coagulation factors.

Objective: Wide interindividual variability exists in response to tissue plasminogen activator (t-PA) treatment in the acute phase of ischemic stroke. We aimed to find genetic variations associated with hemorrhagic transformation (HT) and mortality rates after t-PA. We then generated a clinicalgenetic model for predicting t-PA response. Methods: Our prospective study used SNPlex to genotype 140 single nucleotide polymorphisms (SNPs) from 97 candidate genes in 3 patient cohorts. The cohorts included 1,172 patients who were treated with t-PA; 20.9% of them developed HT as evaluated by systematic brain computed tomography scan, and 10.6% died. A predictive model was generated by logistic regression (LR). Functional studies included real time quantitative polymerase chain reaction, nephelometry, and Western blot for alpha-2-macroglobulin (A2M) and activated partial thromboplastin time measurement for coagulation factor XII (FXII). Results: Replication analysis revealed that the SNP rs669 (Val1000Ile) in A2M was associated with HT, and rs1801020 (-4C>T) of F12 was associated with in-hospital death. The rs669 SNP withstood Bonferroni correction for HT (p < 3.57E-4). LR-based scores predicted HT occurrence (p = 9.13E-15) and in-hospital mortality (p = 8.7E-9) and were validated in an independent cohort. Val1000Ile modified A2M serum levels at baseline and after t-PA infusion, but not mRNA expression or protein structure; -4C>T affected FXII activity both prior to and after t-PA treatment. Interpretation: Two functional polymorphisms were consistently associated with t-PA safety. Our validated LR-based score predicts t-PA safety in the Spanish population. ANN NEUROL 2012;72:716729

SummaryAn elevated concentration of the thrombin-antithrombin complex (TAT) has been associated with high mortality rates and poor outcome in ischaemic stroke patients treated with tissue plasminogen activator (t-PA). Moreover, antithrombin drugs have been tested in combination with t-PA in the acute phase of ischaemic stroke to increase treatment efficacy. We aimed to study whether poor outcome associated with TAT among ischaemic stroke patients treated with t-PA could be due to the effects of this complex on recanalisation rates of the middle cerebral artery (MCA) and on haemorrhagic transformation. The TAT levels of 89 patients having a proximal MCA occlusion were measured by ELISA, and the patients were then treated with t-PA. Complete recanalisation was diagnosed by transcranial Doppler (TCD) at 1, 2 and 6 hours post-t-PA infusion and haemorrhagic transformation was identified by computed tomography (CT). Lower levels of TAT were associated with better recanalisation rates at all time-points (1 hour: OR = 24.8 95% CI 1.4–434.8, p = 0.028;2 hours:OR = 6.3 95% CI 1.5–27, p = 0.014; 6 hours: OR = 6.4 95% CI 1.5–26.5, p = 0.011) after adjustment for stroke risk factors. However, no correlation was found between TAT concentration and haemorrhagic transformation. The elevated mortality rates previously observed in patients with high levels of TAT might have been due to revascularisation resistance. Low levels of TAT are not associated with an increase in haemorrhagic complications after t-PA, indicating that the combination of thrombin blockers and t-PA could be a safe and effective treatment for ischaemic stroke in the future.

Resumen Introduccion La posible existencia de disfuncion endotelial y desequilibrio hemostasico en syndrome X cardiologico, frecuente en la posmenopausia, es un tema controvertido. Objetivo Comparar los biomarcadores: factor de Von Willebrand (FvW), fibrinogeno (Fb), inhibidor del factor tisular total (TFPI-t) y del activador del plasminogeno (PAI-1) en pacientes sin enfermedad arterial coronaria (sindrome X cardiologico, angina mixta y angina vasoespastica) y pacientes con enfermedad arterial coronaria. Material y metodos Se determinaron estos biomarcadores en 10 pacientes con sindrome X cardiologico, en 13 con angina mixta, en 15 con angina vasoespastica, en 10 con enfermedad arterial coronaria y en 20 controles sanos. Resultados Respecto del control sano, todas las pacientes presentaron valores elevados de Fb (3,1 g/l [2,7-3,6] frente a 2,8 g/l [2,2-3,2]; p = 0,03), FvW (211,9% [165-266] frente a 116,5% [91-155]; p Conclusion Los pacientes con sindrome X cardiologico presentan una situacion de disfuncion endotelial y de desequilibrio hemostasico similar a aquellos con angina mixta o angina vasoespastica, aunque menos severo que en pacientes con enfermedad arterial coronaria.

SummaryPlatelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. This study aimed to investigate whether platelets from patients with AMI increase myocardial injury after ischaemia and reperfusion in isolated rat hearts and the modification of this effect by the P2Y12 receptor antagonist cangrelor and the glycoprotein IIb/IIIa receptor blocker abciximab. Isolated rat hearts were subjected to 40 minutes of global ischaemia and 60 minutes of reperfusion. Hearts (four simultaneous experiments per patient) were infused with platelets from nine AMI patients (seven thrombolysed, all on aspirin), untreated or incubated with 10 μM cangrelor or 5 μg/ml abciximab. Control experiments were performed using platelets from healthy volunteers and platelet-poor plasma. P-selectin expression on isolated platelets was higher in AMI patients than in controls and was not modified by the treatments. Control platelets or platelet-poor plasma did mild or no harm. In contrast, platelets from AMI patients significantly augmented myocardial injury, as demonstrated by worse left ventricular (LV) developed pressure, higher maximal LV end-diastolic pressure and coronary resistance, and greater lactate dehydrogenase release and infarct size. Both cangrelor and abciximab greatly attenuated these effects. In conclusion, activated platelets from AMI patients increase myocardial injury after ischaemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment might reduce infarct size by direct effects on reperfused myocardium in these patients.

An unexpected high incidence of thromboembolic complications has been described in patients with systemic autoimmune diseases treated with CD40L immunotherapy. Since activated platelets express CD40L, we aimed to investigate the effects of CD40L mAb in platelet aggregation induced by physiological stimuli. Optical aggregometry was performed on platelet-rich plasma and washed platelets obtained from systemic venous blood (0.38% citrate) of anesthetized pigs. CD40L mAb clone 5c8, used in clinical trials for autoimmune diseases, was used. In platelet-rich plasma, CD40L mAb neither induced platelet aggregation per se, nor significantly affected maximal aggregation or slope of ADP-induced aggregation curves. However, it dose-dependently inhibited spontaneous deaggregation observed in ADP-stimulated samples. This effect was not observed with an irrelevant isotype-matched immunoglobulin. The stabilizing effect on platelet aggregates was neither glycoprotein IIb/IIIa-mediated nor Ca2+-dependent but was abolished by acetylsalicylic acid pretreatment. F(ab')2 fragments did not stabilize ADP-induced platelet aggregates but inhibited the stabilizing effect of CD40L mAb. Similar results were obtained with washed platelets, although higher amplification of ADP-induced aggregation was observed. In conclusion, CD40L expression produced by physiological or pathophysiological platelet activation can sustain a pro-aggregatory effect of CD40L mAb by a mechanism involving mAb Fc domain. These results could help to explain the mechanism of CD40L mAb-induced thromboembolic complications.