Alexander Heiss

The serum protein α2–Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification

Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.

Fetuin-A Regulation of Calcified Matrix Metabolism

The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously in supersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent state in physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcification). Therefore, all tissues not meant to mineralize must be actively protected against chance precipitation of mineral. Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Monomeric fetuin-A protein binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleation cluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineral calciprotein particles. Both monomeric and aggregate forms of fetuin-A mineral accrue acidic plasma protein including albumin, thus stabilizing supersaturated and metastable mineral ion solutions as colloids. Hence, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in a cell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice and humans.

Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A.

Fetuins are serum proteins with diverse functions including the regulation of osteogenesis and inhibition of unwanted mineralization. Besides the alpha2-Heremans and Schmid glycoprotein/fetuin-A, the recently identified fetuin-B is a second member of the fetuin family [Olivier, Soury, Risler, Smih, Schneider, Lochner, Jouzeau, Fey and Salier (1999) Genomics 57, 352-364; Olivier, Soury, Ruminy, Husson, Parmentier, Daveau and Salier (2000) Biochem. J. 350, 589-597], which belongs to the cystatin superfamily. We compared the expressions of fetuin-B and fetuin-A at the RNA level and established that both genes are most highly expressed in liver tissue. Like fetuin-A, fetuin-B mRNA is also highly expressed in tongue and placenta tissues. We demonstrated for the first time that fetuin-B is also expressed at the protein level in sera and several organs of mouse, rat and human. We isolated contiguous genomic clones containing both fetuin-B and fetuin-A genes, indicating that these genes are closely linked at the genome level. The close proximity of both these genes may explain our observation that fetuin-B expression was decreased in fetuin-A-deficient mice. Unlike fetuin-A, the amount of fetuin-B protein in human serum varied with gender and was higher in females than in males. Functional analysis revealed that fetuin-B, similarly to fetuin-A, is an inhibitor of basic calcium phosphate precipitation, albeit less active when compared with fetuin-A. Therefore fetuin-B may have a function that is partly overlapping, if not identical, with the function of fetuin-A.

Hierarchical Role of Fetuin-A and Acidic Serum Proteins in the Formation and Stabilization of Calcium Phosphate Particles

The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.

Clearance of Fetuin-A–Containing Calciprotein Particles Is Mediated by Scavenger Receptor-A

Rationale: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. Objective: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. Methods and Results: In mice, fetuin-A–containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO–deficient mice. Conclusions: We could demonstrate that fetuin-A–containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.

Systemic inhibition of spontaneous calcification by the serum protein alpha 2-HS glycoprotein/fetuin.

The extracellular fluid is a metastable system with regard to calcium and phosphate ions. Active inhibitors of calcification must be present in serum to prevent the spontaneous formation of Ca2+• Pi solid phases which could otherwise precipitate to cause renal calcinosis and block small blood vessels. α2-HS glycoproteins/fetuins, AHSGs, are ideal candidates for this function. AHSGs are ubiquitous and highly abundant in serum; they bind calcium and efficiently prevent de novo formation of apatitic mineral. Normocalcemic AHSG-deficient mice develop sporadic perivascular calcification. Hypercalcemia induced by dietary means or by hormone treatment results in lethal calcinosis in Ahsg−/− mice. A mineral binding structure is proposed for domain D1 of AHSG suggesting that the proposed EF-hand motif for calcium binding does not exist in AHSG. Unlike serum albumin, AHSG does hot preferentially bind ionic Ca2+, but rather in the form of apatitic microcrystals.

A Shielding Topology Stabilizes the Early Stage Protein–Mineral Complexes of Fetuin-A and Calcium Phosphate: A Time-Resolved Small-Angle X-ray Study

Biomineralization mechanisms: The serum protein α2‐HS glycoprotein/fetuin‐A is an important inhibitor that prevents pathological mineralization of calcium phosphate in soft tissues and in the extracellular fluid. TR‐SAXS and stopped‐flow analysis were used to monitor the growth of protein mineral particles nucleating from supersaturated salt solutions in the presence of the protein. It was found that fetuin‐A did not influence the formation of mineral nuclei, but did prevent the aggregation of nuclei and thus mineral precipitation.

Formation and stability kinetics of calcium phosphate–fetuin-A colloidal particles probed by time-resolved dynamic light scattering

Fetuin-A/α2-HS glycoprotein is a major systemic inhibitor of unwanted calcium phosphate deposition in the soft tissue. Fetuin-A mediates the formation of initially ∼100 nanometre sized colloidal protein–mineral particles, denoted as calciprotein particles. After a lag period, these particles rapidly grow to elongated particles of about twice the initial size. Similar particles can be generated with other acidic macromolecules as well. However, fetuin-A is more potent regarding activity and specificity of inhibition. Given the widely recognized physiological relevance of fetuin-A as an inhibitor of pathological mineralization both in vitro, in cells, in animals and in humans, and its ready availability in large quantities, fetuin-A is a prototypic model protein to investigate protein–calcium phosphate interactions and mineral colloid stabilization, respectively. Several studies have concentrated on the pathophysiological relevance and the structural details of particle formation and mineral ripening. The principles governing calciprotein particle formation and ripening are nevertheless poorly understood. Here we present a systematic and quantitative investigation by time-resolved dynamic light scattering of the three major parameters: fetuin-A concentration, mineral ion concentration and temperature. Changes in temperature had only a weak effect on calciprotein particle size. Increased mineral ion concentrations and especially increased fetuin-A concentration led to smaller particles. An increased temperature, mineral ion concentration and a reduced fetuin-A concentration, respectively, all accelerated the particle ripening process. Our investigation demonstrates that calciprotein particle formation and ripening are two separate and successive processes and that particle ripening follows Arrhenius law. Furthermore it provides concepts to control the particle size and stability.

Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification

Objective— The drug warfarin blocks carboxylation of vitamin K–dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Approach and Results— Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor–dependent and PS-dependent manner. Conclusions— Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors.