Induced forms of α2-macroglobulin neutralize heparin and direct oral anticoagulant effects.

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.

Effect of α2­macroglobulin in the early stage of jaw osteoradionecrosis.

Advanced osteoradionecrosis (ORN) is one of the most serious complications in patients with head and neck cancer, resulting in poor prognosis. Numerous studies have therefore focused on the pathogenesis and interventions of ORN early stage. The present study aimed to investigate whether α2­macroglobulin (α2M) could prevent early­stage jaw osteoradionecrosis caused by radiotherapy (RT). Following local injection of α2M, a single dose of 30 Gy was delivered to rats for pathological exploration. For 28 days, the irradiated mandible and soft tissues were examined for potential changes. Furthermore, primary human bone marrow mesenchymal stem cells pretreated with α2M followed by 8 Gy irradiation (IR) were also used. Tartrate­resistant acid phosphatase assay, terminal uridine deoxynucleotidyl nick end labeling assay and immunohistochemical staining were performed on irradiated mandibular bone, tongue or buccal mucosa tissues from rats. Cell proliferation was assessed by evaluating the cell morphology by microscopy and by using the cell counting kit­8. Fluorescence staining, flow cytometry and western blotting were conducted to detect the reactive oxygen species level, cell apoptosis and protein expression of superoxide dismutase 2 (SOD2), heme oxygenase­1 (HO­1) and phosphorylated Akt following irradiation. The results demonstrated that α2M attenuated physical inflammation, osteoclasts number and fat vacuole accumulation in mandibular bone marrow and bone marrow cell apoptosis following IR in vivo. Furthermore, α2M pretreatment suppressed the expression of 8­hydroxy­2'­deoxyguanosine in mandibular bone and tongue paraffin embedded sections, which is a marker of oxidative damage, and increased SOD2 expression in mucosa and tongue paraffin embedded sections. The present study demonstrated the efficient regulation of antioxidative enzymes, including SOD2 and heme oxygenase­1, and reduction in oxidative damage by α2M. In addition, in vitro results confirmed that α2M may protect cells from apoptosis and suppress reactive oxygen species accumulation. Overall, the present study demonstrated that α2M treatment may exert some radioprotective effects in early­stage ORN via antioxidant mechanisms, and may therefore be considered as a potential alternative molecule in clinical prophylactic treatments.

Neurogenic Thoracic Outlet Syndrome and other Forms of Cervical Brachial Syndrome Treated with Plasma Concentrate Enriched for Alpha 2 Macroglobulin.

BACKGROUND: Existing therapies for myofascial and neuralgic forms of cervicobrachial pain may have unsatisfactory outcomes. Alternative therapies may be considered, particularly for individuals who have failed to respond. Contemporary conceptualizations of chronic pain mechanisms include the contribution of inflammatory factors; therefore, locally targeted antiinflammatory administrations may play a role in treatment of cervicobrachial pain.Alpha 2 macroglobulin (A2M) is a plasma protein that acts as a molecular trap for inflammatory factors such as tumor necrosis factor. After plasma is enriched for A2M, it may be considered as a possible injectable agent to counteract inflammation that may occur with a cervicobrachial pain syndrome. OBJECTIVES: This retrospective review evaluates patient response to the use of plasma concentrate enriched for alpha 2 macroglobulin (A2M-PPP) in treatment of neurogenic thoracic outlet syndrome (TOS) and other forms of cervical brachial syndrome. STUDY DESIGN: Observational Study. SETTING: Outpatient interventional neurology practice. METHODS: There were 62 patients, including 46 women and 16 men ages 23-77 years. Twenty-three of these patients were diagnosed with complex regional pain syndrome (CRPS) or fibromyalgia, 18 with TOS, and 21 with musculotendinous pain (MTP). At baseline, 1 month, 3 months, and 6 months, patient status was evaluated with a Brief Pain Inventory (BPI) that included a composite pain score and a functional interference score. Patients were asked to estimate overall satisfaction with a Patient Global Impression of Change (PGIC) scale. Criterion for clinically significant improvement included >30% betterment in the BPI pain and functional interference subscales and a PGIC of > 5 at the 3-month mark. RESULTS: Three patients, one with CRPS and 2 with TOS, complained of several days of worsened pain or dysesthesias. No serious or permanent complications were encountered. For patients with TOS at the 3-month mark, 61% achieved clinical endpoints of success compared with 35% with CRPS/fibromyalgia and 24% for patients with MTP (P < 0.05, chi-square). By 6 months, 22% of individuals in the neuropathic TOS group had > 30% improvements in pain and functional interference scores compared with 13% of the individuals in the CRPS/fibromyalgia group and 18% in the MTP group. LIMITATIONS: This article does not differentiate the added benefit of A2M-PPP from hydrodissection alone. Additionally, this article does not evaluate the actual benefit of the A2M molecule apart from other factors present in the platelet-poor concentrate such as exosomes and cytokines. With the advent of pure engineered A2M, more focused studies will be possible. Also, an independent assay was not done, and therefore we cannot be precisely sure about the exact quantity of platelets, if any, which were contained in the platelet-poor concentration. CONCLUSIONS: Results suggest that A2M-PPP, when injected into muscle, tendon, and epineurium with live ultrasound guidance, appears to be relatively safe and free of postinjection inflammatory reactions that are often seen after platelet-poor plasma injection. A2M-PPP appears to be associated more frequently with good outcomes when injected into brachial plexus targets in patients with TOS compared with outcomes observed after injection of the plexus in patients with CRPS/fibromyalgia. KEY WORDS: Plasma concentrate enriched for alpha 2 macroglobulin, neurogenic thoracic outlet syndrome, cervical brachial syndrome.

Early supplemental α2-macroglobulin attenuates cartilage and bone damage by inhibiting inflammation in collagen II-induced arthritis model.

OBJECTIVE: To determine if early supplemental intra-articular α2-macroglobulin (A2M) has a chondroprotective effect in a collagen II-induced arthritis (CIA) mice model. METHODS: DBA/1 mice were randomized into four groups (n = 15/group): (a) CIA + 1.2 µg of A2M; (b) CIA + 0.8 µg of A2M; (c) CIA + 0.4 µg of A2M; (d) vehicle + phosphate-buffered saline (PBS). A2M was injected into right ankles and PBS was injected into the left ankles simultaneously as internal control at days 36, 43 and 50. The CIA inflammation clinical score and ankle thickness were recorded every other day starting on day 21 until sacrifice. Changes in inflammation were monitored by in vivo fluorescence molecular tomography (FMT). Inflammation, cartilage and bone damage were assessed with X-ray, histology and immunohistochemistry. Cartilage and inflammation-related gene expression was quantified by real-time polymerase chain reaction (PCR). RESULTS: All mice showed ankle inflammation on day 33. After day 43, lower clinical scores, ankle thickness and Sharp/van der Heijde method scores in A2M-treated ankles compared with PBS-treated ankles. FMT data indicated that the inflammation markers MMPSense and ProSense were significantly elevated in the PBS-treated ankles than A2M-treated ankles. Histology and X-ray analyses indicated that A2M administration resulted in lower levels of inflammatory infiltration and synovial hyperplasia, as well as more typical cartilage and bone organization with increased COL II and Aggrecan staining when compared with PBS-treated ankles. In addition, real-time PCR showed that,matrix metalloproteinase-3, -9, -13, COL X and Runx2 were significantly less expressed in A2M-treated groups than PBS-treated animals. CONCLUSION: Early supplemental intra-articular A2M exerts an anti-inflammatory effect and attenuates cartilage and bone damage in a CIA model.

Alpha 2-Macroglobulin as Dual Regulator for Both Anabolism and Catabolism in the Cartilaginous Endplate of Intervertebral Disc.

STUDY DESIGN: Basic science study. OBJECTIVE: To illustrate supplemental alpha-2 macroglobulin (α2 M) has beneficial effects on cartilaginous endplates (CEPs) that may slow the progression of intervertebral disc (IVD) degeneration. SUMMARY OF BACKGROUND DATA: CEPs play a vital role in progression of intervertebral disc degenerative diseases. However, the ideal and economic therapies for CEPs degeneration are still urgently required. METHODS: Firstly, we confirmed degenerative CEP characters by H&E and Safranin O fast green staining and detected increasing level of α2 M and matrix metalloproteinase 13(MMP-13) in degenerative CEP by immunohistochemistry. Then, effects of exogenous α2 M on tumor necrosis factor alpha (TNF-α)-induced CEP catabolic enzyme and anabolic molecules were evaluated by qRT-PCR, Western blotting and ELISA in cultured CEP cells obtained from rats. Furthermore, suppression of α2 M on TNF-α-induced activation of NF-кB signaling pathway was measured by Western blotting and immunofluorescence. In addition, function of α2 M on TNF-α-treated ex vivo IVDs from rats lumbar IVDs was estimated by measuring the expression of MMP-13, Sox9, aggrecan, and type II collagen in CEP area. RESULTS: Compared with normal CEP, level of α2 M was slightly increased in CEP from degenerative patients, whereas MMP-13 was sharply elevated. In vitro, α2 M inhibited expression and activity of MMP-3 or MMP-13 in a dose-dependent manner in rat CEP cells stimulated by TNF-α. The α2 M refrained phosphorylation of IκBα and inhibited nuclear translocation of p65. Finally, supplemental α2 M reduced expression of MMP-13, and promoted expression of Sox9, aggrecan, and type II collagen in CEP area of ex vivo IVDs cultured with TNF-α. CONCLUSION: α2 M is not sufficiently produced to inactivate higher concentrations of catabolic factor MMP-13 found in the degenerated CEP. Supplemental α2 M protects against the progression of IVD degeneration by inhibiting effects of proinflammatory cytokines. LEVEL OF EVIDENCE: N/A.

Protective role of α2-macroglobulin against jaw osteoradionecrosis in a preclinical rat model.

OBJECTIVE: We have previously demonstrated the effect of alpha-2-macroglobulin (α2M) in the remediation of radiation-induced cellular damage. Here, we investigated the protective effects of α2M in a preclinical rat model of jaw osteoradionecrosis (ORN). METHODS: Eighteen rats were divided randomly into three groups: the control group, the radiation therapy (RT) alone group, and the radiated mandibles pretreated with α2M (α2M + RT) group. One month after radiation, all left molar teeth were extracted. After another 3 months, the animals were sacrificed and body weight, histopathology, microcomputed tomography and immunofluorescence were evaluated in all groups. RESULTS: The RT group showed serious alopecia, bone exposure, inflammation, necrosis, fibrosis, and the absence of new bone formation within the socket. The α2M + RT group exhibited less alopecia than the RT group and slight inflammation and fibrosis in the bone marrow cavity. The cortical bone was similar to normal bone tissue. Interestingly, compared with RT group, serum superoxide dismutase levels in the α2M + RT group increased at the 1th day (P = 0.037), 14th day (P = 0.012), while reactive oxygen species levels clearly decreased at the 1th day (P< 0.001), 14th day (P = 0.007), and 28th day (P = 0.013). CONCLUSIONS: A clinically translational model of jaw ORN was successfully established and the application of α2M prior to radiation protected the bone from being injured by the radiation, possibly related to oxidative stress.