Ran Ni

Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice.

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2(-/-) mouse. As in GPS, Nbeal2(-/-) mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2(-/-) platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2(-/-) platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2(-/-) bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2(-/-) mice has deleterious effects on megakaryocyte survival, development, and platelet production.

The maternal immune response to fetal platelet GPIbα causes frequent miscarriage in mice that can be prevented by intravenous IgG and anti-FcRn therapies

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal/neonatal platelets. It is the most common cause of severe thrombocytopenia in neonates, but the frequency of FNIT-related miscarriage is unknown, and the mechanism(s) underlying fetal mortality have not been explored. Furthermore, although platelet αIIbβ3 integrin and GPIbα are the major antibody targets in immune thrombocytopenia, the reported incidence of anti-GPIbα-mediated FNIT is rare. Here, we developed mouse models of FNIT mediated by antibodies specific for GPIbα and β3 integrin and compared their pathogenesis. We found, unexpectedly, that miscarriage occurred in the majority of pregnancies in our model of anti-GPIbα-mediated FNIT, which was far more frequent than in anti-β3-mediated FNIT. Dams with anti-GPIbα antibodies exhibited extensive fibrin deposition and apoptosis/necrosis in their placentas, which severely impaired placental function. Furthermore, anti-GPIbα (but not anti-β3) antiserum activated platelets and enhanced fibrin formation in vitro and thrombus formation in vivo. Importantly, treatment with either intravenous IgG or a monoclonal antibody specific for the neonatal Fc receptor efficiently prevented anti-GPIbα-mediated FNIT. Thus, the maternal immune response to fetal GPIbα causes what we believe to be a previously unidentified, nonclassical FNIT (i.e., spontaneous miscarriage but not neonatal bleeding) in mice. These results suggest that a similar pathology may have masked the severity and frequency of human anti-GPIbα-mediated FNIT, but also point to possible therapeutic interventions.

Diannexin, an annexin A5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation

Background:  Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life.

BF061, a novel antiplatelet and antithrombotic agent targeting P2Y12 receptor and phosphodiesterase

The addition of phosphodiesterase (PDE) inhibitors has been reported to potentiate the antithrombotic effects of P2Y₁₂ antagonists without increasing bleeding risk. In this study, we report that a potent antiplatelet agent, 2-ethylthio-6-phenethylaminoadenosine (BF061), inhibits platelet activation and thrombosis via P2Y₁₂ antagonism and PDE inhibition. We explored the antiplatelet mechanism of BF061 by measuring cAMP, cGMP levels, PDE activity, and the interaction between ADP and P2Y₁₂ using atomic force microscopy. The antithrombotic effect of BF061 was evaluated in mice using intravital microscopy in FeCl₃₋induced mesenteric and laser-induced cremasteric arterial thrombosis models. BF061 robustly inhibited platelet aggregation and ATP release induced by multiple platelet agonists via P2Y₁₂ antagonism and PDE inhibition. Interestingly, despite being structurally similar to BF061, P2Y₁₂ receptor antagonist AR-C69931MX had no effect on human platelet PDE. In FeCl3-induced mesenteric arterial thrombosis model, BF061 effectively prevented thrombus formation similarly to clopidogrel; it also reduced thrombus volume in laser-injured cremaster arteriole model. In contrast, BF061 induced dramatically less bleeding at an antithrombotic dose compared to clopidogrel. In summary, we developed a novel antiplatelet and antithrombotic agent targeting both P2Y₁₂ and PDE. Given the prevalence of combined antiplatelet therapy in clinical practice, an antiplatelet agent bearing dual activities may have therapeutic advantage as a potential antithrombotic drug.

Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein

Factor (F) XII, a key component of the contact system, triggers clotting via the intrinsic pathway, and is implicated in propagating thrombosis. Although nucleic acids are potent activators, it is unclear how the contact system is regulated to prevent uncontrolled clotting. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa and attenuates its capacity to trigger coagulation. To investigate the role of HRG as a regulator of the intrinsic pathway, we compared RNA- and DNA-induced thrombin generation in plasma from HRG-deficient and wild-type mice. Thrombin generation was enhanced in plasma from HRG-deficient mice, and accelerated clotting was restored to normal with HRG reconstitution. Although blood loss after tail tip amputation was similar in HRG-deficient and wild-type mice, carotid artery occlusion after FeCl3 injury was accelerated in HRG-deficient mice, and HRG administration abrogated this effect. To confirm that HRG modulates the contact system, we used DNase, RNase, and antisense oligonucleotides to characterize the FeCl3 model. Whereas DNase or FVII knockdown had no effect, carotid occlusion was abrogated with RNase or FXII knockdown, confirming that FeCl3-induced thrombosis is triggered by RNA in a FXII-dependent fashion. Therefore, in a nucleic acid-driven model, HRG inhibits thrombosis by modulating the intrinsic pathway of coagulation.

Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models.

Haemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 - 0.27, 0.08 - 0.20, and 0.03 - 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 μg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 μg/ml, respectively) (95% CI = 2.4 - 20, 0.05 - 0.33, and 0.06 - 2.2 μg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 μg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

Atorvastatin Delays Murine Platelet Activation In Vivo Even in the Absence of Endothelial NO Synthase

Objective—Statins decrease mortality in patients with vascular disorders, and evidence for the pleiotropic effects of statins is accumulating. Statins enhance endothelial NO synthase (eNOS) expression, thereby attenuating platelet activation and thrombus formation. Our goal was to determine whether statins have eNOS-independent effects on platelet activation. Methods and Results—Wild-type and eNOS-deficient mice were given a 14-day course of oral atorvastatin, and platelet activation was evaluated in vitro and in vivo. Whereas in wild-type mice atorvastatin inhibited platelet activation in vitro in response to numerous agonists, in eNOS-deficient mice, atorvastatin inhibited only thrombin-induced and protease-activated receptor 4 agonist peptide–induced platelet activation. Consistent with an eNOS-independent effect, atorvastatin inhibited platelet activation in vivo in both wild-type and eNOS-deficient mice. Conclusion—Atorvastatin inhibits platelet activation via eNOS-dependent and eNOS-independent mechanisms with the latter restricted to protease-activated receptor 4-induced activation downstream to the receptor.

Zinc delays clot lysis by attenuating plasminogen activation and plasmin-mediated fibrin degradation

Zinc circulates free in plasma at a concentration of 0.1-2 µM, but its levels increase locally when it is released from activated platelets. Although zinc influences many processes in haemostasis, its effect on fibrinolysis has not been thoroughly investigated. Using a fluorescent zinc-binding probe, we demonstrated that zinc binds tissue-type plasminogen activator (tPA) and plasmin with high affinity (Kd values of 0.2 µM), and surface plasmon resonance studies revealed that zinc binds fibrin with a Kd of 12.8 µM. Zinc had no effect on the affinity of plasminogen or plasmin for fibrin, but increased the affinity of tPA by two-fold. In the presence of 5 µM zinc, the catalytic efficiency of plasminogen activation by tPA was reduced by approximately two-fold, both in the absence or presence of fibrin. Zinc attenuated plasmin-mediated degradation of the fibrinogen alpha-chain by 43 %, but had no effect on trypsin degradation. tPA-mediated fibrin clot lysis was prolonged 2.5-fold by zinc in a concentration-dependent fashion, and tPA-mediated plasma clot lysis was attenuated by 1.5-fold. Therefore, our data indicate that zinc modulates fibrinolysis by attenuating tPA-mediated plasminogen activation and plasmin-induced fibrin degradation. These findings suggest that local release of zinc by platelets attenuates fibrinolysis.

Comparison of the effect of dabigatran and dalteparin on thrombus stability in a murine model of venous thromboembolism.

Essentials Does thrombus stability alter the presentation of venous thromboembolism and do anticoagulants alter this? In a murine model, we imaged a femoral vein thrombus and quantified emboli in the pulmonary arteries. Dabigatran decreases thrombus stability via factor XIII increasing embolization and pulmonary emboli. This cautions against the unapproved use of dabigatran for acute initial treatment of deep vein thrombosis.

Rosuvastatin Reduces Aortic Sinus and Coronary Artery Atherosclerosis in SR-B1 (Scavenger Receptor Class B Type 1)/ApoE (Apolipoprotein E) Double Knockout Mice Independently of Plasma Cholesterol Lowering

Objective— Rosuvastatin has been widely used in the primary and secondary prevention of coronary heart disease. However, its antiatherosclerotic properties have not been tested in a mouse model that could mimic human coronary heart disease. The present study was designed to test the effects of rosuvastatin on coronary artery atherosclerosis and myocardial fibrosis in SR-B1 (scavenger receptor class B type 1) and apoE (apolipoprotein E) double knockout mice. Approach and Results— Three-week-old SR-B1−/−/apoE−/− mice were injected daily with 10 mg/kg of rosuvastatin for 2 weeks. Compared with saline-treated mice, rosuvastatin-treated mice showed increased levels of hepatic PCSK9 (proprotein convertase subtilisin/kexin type-9) and LDLR (low-density lipoprotein receptor) message, increased plasma PCSK9 protein but decreased levels of hepatic LDLR protein and increased plasma total cholesterol associated with apoB (apolipoprotein B) 48-containing lipoproteins. In spite of this, rosuvastatin treatment was associated with decreased atherosclerosis in both the aortic sinus and coronary arteries and reduced platelet accumulation in atherosclerotic coronary arteries. Cardiac fibrosis and cardiomegaly were also attenuated in rosuvastatin-treated SR-B1−/−/apoE−/− mice. Two-week treatment with rosuvastatin resulted in significant decreases in markers of oxidized phospholipids in atherosclerotic plaques. In vitro analysis showed that incubation of bone marrow-derived macrophages with rosuvastatin substantially downregulated cluster of differentiation (CD)36 and inhibited oxidized LDL-induced foam cell formation. Conclusions— Rosuvastatin protected SR-B1−/−/apoE−/− mice against atherosclerosis and platelet accumulation in coronary arteries and attenuated myocardial fibrosis and cardiomegaly, despite increased plasma total cholesterol. The ability of rosuvastatin to reduce oxidized phospholipids in atherosclerotic plaques and inhibit macrophage foam cell formation may have contributed to this protection.