Alpha-2-macroglobulin as an equine joint therapeutic

Alpha-2-macroglobulin (A2M) is an abundant native blood glycoprotein found across all mammalian species, including horses. Its unique conformation, of four identical subunits comprising a large tetramer, allows A2M to act as a broad-spectrum protease inhibitor. Proteases, or enzymes that break down proteins, cleave a specific peptide sequence in A2M’s bait region leading to a conformation change, entrapping the protease away from its target (Figure 1).¹

This bait-and-trap mechanism is responsible for A2M’s anti-protease activity, and has been demonstrated to have an anti-inflammatory and cartilage-protective effect in benchtop and rodent laboratory models.^2–4 Recently, A2M has been shown to be highly abundant in equine joints with osteoarthritis (OA), compared to normal joints.⁵ More research is needed to understand the role of A2M’s innate upregulation in OA as currently, it is unknown if A2M’s upregulation in the joint is a homeostatic and compensatory mechanism to combat joint inflammation or a byproduct of the disease state.

Current evidence supports the use of orthobiologics, which are prepared from patients’ whole blood for intra-articular delivery, to concentrate A2M. This allows for the delivery of A2M to inflamed target tissues, mainly the synovium, as a non-pharmacologic joint therapy. There, A2M can act to inhibit the proteases (ex: matrix metalloproteinases or MMPs) which propagate synovial inflammation and cyclical cartilage degradation central to progressive joint conditions such as OA.

While a clinician’s choice of intra-articular blood-based joint therapy is multifaceted, patient-specific, and could be expanded into a standalone article, A2M orthobiologics are particularly useful in cases where an acellular, concentrated product is of clinical utility. Briefly, indications include synovitis in its early or advanced stages, cartilage damage, radiographic OA, or cases of acute or repetitive trauma where cartilage protection from inflammatory mediators would be of clinical merit. Importantly, A2M may pose a safer alternative to corticosteroids for horses with endocrinopathies such as pituitary pars intermedia dysfunction or insulin dysregulation, where steroid-associated laminitis may be of high concern.⁶

All blood-based orthobiologics contain A2M. The relative A2M abundance depends on the specific processing involved for each commercially available kit.⁷ The current equine A2M orthobiologic on the market in the United States is Alpha2EQ by Creative Sciences. In contrast to products like platelet-rich-plasma, autologous conditioned serum, and autologous protein solution which rely on platelet or cellular function, the Alpha2EQ product removes cells and platelets in its first centrifugation step. Furthermore, it differs from autologous conditioned serum and protein products in that there is no ‘conditioning’ step, meaning that it does not contain beads which prime the blood components to release additional growth factors or cytokines. Rather, the conical tube used in the second centrifugation step contains a filter which retains large proteins in the injectate, while excluding small proteins in the fraction which is discarded (Figure 2).

Like other orthobiologics, multiple joints may be treated in one session, without concerns for complications that could accompany multi-joint injections with corticosteroids. Due to the large and complex nature of A2M and lack of species-specific reagents, researchers currently face problems with evaluation of this equine glycoprotein across all orthobiologic products. There is no reliable ELISA or straightforward benchtop assay to quantify concentration of the glycoprotein, and work is ongoing to develop other analytical methods for equine A2M.^5,8 Untargeted proteomic methods, such as mass spectrometry, offer a broad but intensive analysis of all proteins contained in the final products for injection. This type of analysis could reveal other proteins important in the mechanism of A2M-containing orthobiologics and provide a more complete picture of why they demonstrate a beneficial clinical effect in our equine patients.

Another important consideration of any product prepared from the patient for autologous injection are potential effects of patient factors on orthobiologic composition. The final concentration of cells, proteins, and cytokines is known to affect patient outcome, and so a complete understanding of factors influencing composition is critical as we move forward using orthobiologics in our diverse patients. A variety of patient factors such as systemic health, dehydration, exercise status, medications or vaccines administered, and pituitary pars intermedia dysfunction are currently under investigation for their impact on A2M orthobiologics and other autologous blood-based joint therapies.^9–11

To date, there is evidence illustrating that A2M orthobiologics, prepared from humans and from horses, have anti-protease and anti-inflammatory effects.^12,13 In inflamed synovial cells collected from equine joints, cell-culture treatment with the commercially available equine A2M orthobiologic resulted in downregulation of proinflammatory genes known to be important in the progression of OA, supporting an anti-inflammatory action distinct from the classic “bait-and-trap” mechanism.¹³ A recent exploratory study in horses with chronic degenerative joint disease showed that the European A2M orthobiologic reduced inflammatory mediator concentrations in joints 30 days after intra-articular treatment.¹⁴

While research is ongoing to characterize A2M as an emerging orthobiologic treatment, current evidence supports its continued use as an anti-inflammatory joint therapy. In blood-based orthobiologics, A2M is likely a singular, potent, synergistic component in a milieu of innate bioactive factors which act to reduce joint inflammation with associated pain and lameness in horses. As clinicians continue to experience positive clinical outcomes for their patients treated with A2M-containing products, there is still much to be uncovered before we understand how each product exerts its beneficial intra-articular effects.

References

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2. Wang S, Wei X, Zhou J, et al. Identification of α2‐Macroglobulin as a Master Inhibitor of Cartilage‐Degrading Factors That Attenuates the Progression of Posttraumatic Osteoarthritis. Arthritis Rheumatol. 2014;66(7):1843-1853. doi:10.1002/art.38576
3. Luan Y, Kong L, Howell DR, et al. Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin. Osteoarthr Cartil. 2008;16(11):1413-1420. doi:10.1016/j.joca.2008.03.017
4. Tortorella MD, Arner EC, Hills R, et al. α2-Macroglobulin Is a Novel Substrate for ADAMTS-4 and ADAMTS-5 and Represents an Endogenous Inhibitor of These Enzymes*. J Biol Chem. 2004;279(17):17554-17561. doi:10.1074/jbc.m313041200
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8. Ortved KF, Alward L, Cowles B, et al. Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices. Front Vet Sci. 2024;11:1335972. doi:10.3389/fvets.2024.1335972
9. Rinnovati R, Romagnoli N, Gentilini F, Lambertini C, Spadari A. The influence of environmental variables on platelet concentration in horse platelet-rich plasma. Acta Vet Scand. 2016;58(1):45. doi:10.1186/s13028-016-0226-3
10. Hale JN, Hughes KJ, Hall S, Labens R. The effect of exercise on cytokine concentration in equine autologous conditioned serum. Equine Vet J. 2023;55(3):551-556. doi:10.1111/evj.13586
11. Moorman VJ, Gordon J, Page AE, Adams AA, Hart KA. Cytokine profiles of autologous protein solution (APS) and autologous conditioned serum (ACS) from horses with pituitary pars intermedia dysfunction (PPID) and healthy horses: A preliminary investigation. J Equine Rehabilitation. 2025;3:100039. doi:10.1016/j.eqre.2025.100039
12. Showel KK, Evans CH, Vega RED la, et al. Preparation of alpha-2 macroglobulin–rich plasma from human blood—technique and compositional analysis of plasma, platelet-rich plasma, platelet-poor plasma, and alpha-2 macroglobulin–rich plasma. The Journal of Cartilage and Joint Preservation. 2025;5(4). doi:10.1016/j.jcjp.2025.100255
13. Pugliese BR, Rosario FKR, Schnabel LV. Alpha2EQ downregulates proinflammatory cytokine and chemokine gene expression in cultured synovial fibroblasts. Am J Vet Res. 2025:1-12. doi:10.2460/ajvr.25.09.0319
14. Gugliandolo E, Biondi V, Luca MD, et al. Pilot Study of an Alpha-2-Macroglobulin-Enriched Plasma-Derived Orthobiologic Preparation in Sport Horses with Chronic Degenerative Joint Disease. Vet Sci. 2026;13(6):536. doi:10.3390/vetsci13060536