Nima Vaezzadeh

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.

Tissue Factor and Thrombosis Models

Mouse models of thrombosis have extended our understanding of the role of tissue factor (TF) in thrombogenesis. Because tissue factor deficiency is embryonic lethal in mice, inventive genetic models are required to probe the role of TF in thrombosis. TF is expressed by different cell types, including vascular smooth muscle cells, cardiomyocytes, fibroblasts, and monocytes. Platelets and endothelial cells also express TF under certain conditions, but the importance of this TF remains controversial. Animal models are commonly used to evaluate the contribution of TF from each cell type to thrombogenesis. Although a variety of well-established injury techniques are used to induce thrombosis, it is likely that the sources of TF that drive thrombosis are model dependent. Therefore, rigorous controls are needed before thrombogenesis can be attributed to TF from a particular cell type. This review summarizes data from mouse models that have attempted to delineate the role of TF in thrombus formation in response to various types of vascular injury. We have consolidated this information to generate unifying concepts that require testing in future studies.

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.

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.

Tissue factor microparticles and haemophilia

Treatment choices for haemophilia patients with inhibitors are suboptimal. Tissue factor-bearing microparticles home to thrombi in a cell adhesion molecule-dependent fashion. Their potential utility as a procoagulant is discussed along with the challenges of evaluating this approach in mouse models of haemophilia.

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.

Targeted Knockdown of Tissue Factor in B16F10 Melanoma cells suppresses their Ability to Metastasize to Bone and cause cancellous Bone Loss

In this study, we use a well-defined mouse model to examine tissue factor’s (TF) role in osteolytic bone metastasis. C57BL/6 mice received either mock siRNA-transfected or TF-specific siRNA-transfected B16F10 melanoma cells by left ventricular injection. A third group served as an age-matched control and did not receive any tumour cells. The effect on tumour burden and bone strength was then determined 14 days later by using bone histomorphometry and biomechanical testing. Based on histomorphometric analysis of the femurs, mice receiving TF-specific siRNA-transfected tumour cells had significantly reduced tumour burden as compared to those from mice that received mock siRNA-transfected tumour cells (2.20 ± 0.58% vs. 9.18 ± 2.20%). Furthermore, the femurs from mice receiving TF siRNA-transfected tumour cells displayed decreased osteoclast surface and consequently, increased cancellous bone volume and strength when compared to those isolated from mice that were injected with mock-transfected tumour cells. More importantly, no differences in osteoclast surface or cancellous bone volume and strength were observed when the femurs of mice that received TF siRNA-transfected tumour cells were compared to control mice that did not receive tumour cells. Based on these findings, we conclude that the expression of TF by tumour cells promotes their ability to metastasize to bone, thereby facilitating tumour cell—induced cancellous bone loss.