Background Cancer patients have an approximately four-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF+) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF+ tumor-derived MVs (TMVs) activate platelets in vitro and in mice. Materials and Methods We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF+ TMVs on platelets and thrombosis. Results and Conclusions We found that both types of TF+ TMVs activated human platelets and induced aggregation in vitro in a TF- and thrombin-dependent manner. Further, injection of BxPc-3 TF+ TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF+ TMV-induced platelet activation contributes to thrombosis in cancer patients.
Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes, remains incompletely understood. Prior studies suggest that megakaryocytes shed platelets in the pulmonary vasculature. To better understand thrombopoiesis and to develop a potential platelet transfusion strategy that is not dependent upon donors, of which there remains a shortage, we examined whether megakaryocytes infused into mice shed platelets. Infused megakaryocytes led to clinically relevant increases in platelet numbers. The released platelets were normal in size, displayed appropriate surface markers, and had a near-normal circulating half-life. The functionality of the donor-derived platelets was also demonstrated in vivo. The infused megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers. IntroductionWhile the number of platelet donors is increasing, there is still a significant donor shortage due to the growing population of patients with serious illnesses associated with thrombocytopenia and hemorrhage (1). The use of donor-derived platelets raises the following concerns: variability of quality and quantity, risk of infectious transmission, short lifespan of stored platelets, bacterial contamination during storage, and development of alloantibodies in multi-transfused patients. These problems highlight a need for new strategies to generate platelets for infusion therapy. Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes remains incompletely understood. In vitro studies suggest that platelets form nodes at tips of proplatelet strands (2). However, direct visualization of live calvaria marrow using multiphoton intravital microscopy suggests that megakaryocytes release large cytoplasmic fragments into the vasculature (3), which must then undergo reorganization into platelets. Studies based on morphologic analysis and quantification of megakaryocyte-like polyploid nuclei in the pulmonary venous system suggested that megakaryocytes release platelets in the lungs (4). Derivation of platelets from megakaryocytes in culture was first reported in 1995 (5) but has been difficult to quantitatively upscale. To date, the best published result from infused in vitro produced platelets used irradiated mice with low platelet counts (~10 4 /μl) (6). Peak percent donor platelet counts were still only 1%-2%. Given the limited success by which platelets have been generated ex vivo, we examined whether infused megakaryocytes release platelets in vivo. We found that by infusing ex vivo generated murine megakaryocytes into mice, we can achieve an approximately 100-fold increase in recipient platelet count over prior published results, achieving clinically relevant levels of donor platelets. These platelets have a slightly shorter
• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)
Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo–generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo–generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo–generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo–derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.
We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human ␣IIb3. RUC-1 did not inhibit ␣V3, suggesting that it interacts with ␣IIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1's effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the ␣IIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid ␣IIb3 receptor composed of human ␣IIb and murine 3, but not a hybrid receptor composed of murine ␣IIb and human 3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid h␣IIb/m3 receptors. Collectively, these data support RUC-1's specificity for ␣IIb, provide new insights into the ␣IIb binding pocket, and establish RUC-1's antithrombotic effects in vivo. (Blood. 2009;114:195-201) IntroductionWe previously published data on the identification of a novel inhibitor of ␣IIb3 (Compound 1; now referred to as RUC-1). 1 We speculated that it interacted exclusively with the ␣IIb portion of the Arg-Gly-Asp (RGD) binding site based on its specificity for ␣IIb3 compared with ␣V3 and molecular docking studies into the human ␣IIb3 headpiece suggesting that the positively charged piperazinyl nitrogen of RUC-1 interacts with the carboxyl group of D224 in ␣IIb and that the heterocyclic fused ring of RUC-1 interacts with one or more of the 3 aromatic residues that line the ␣IIb pocket. RUC-1 also is too short to span between D224 of ␣IIb and the 3 metal ion-dependent adhesion site (MIDAS) and lacks a carboxyl group to coordinate the MIDAS metal ion, which is an invariant feature of all other small molecule ␣IIb3 antagonists. [2][3][4] In the present study, we further tested whether RUC-1 demonstrates specificity for ␣IIb by taking advantage of known differences in the abilities of ␣IIb3 antagonists to inhibit ␣IIb3-mediated platelet aggregation in different species. Consistent with these data, we also found that RUC-1 could inhibit thrombus formation in vivo in transgenic mice expressing human (h) ␣IIb in complex with murine (m) 3, but not wild-type (WT) mice. Estimates of electrostatic and van der Waals interaction energies of RUC-1 docked into the crystal structure of human ␣IIb3 or molecular models of rat ␣IIb3, mouse ␣IIb3, or hybrid human ␣IIb/mouse3 were consistent with the functional data. In aggregate, these data have important implications for understanding the structure of the ␣IIb binding pocket and the potential antiplatelet effects of ␣IIb-specific ␣IIb3 antagonists. Me...
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