Clinical and Investigative Medicine

 

Multimerin: a bench-to-bedside chronology of a unique platelet and endothelial cell protein from discovery to function to abnormalities in disease

Catherine P.M. Hayward, MD, PhD

Clin Invest Med 1997;20(3):176-187

[résumé]

Part of this work was presented as the 1996 Gold Medal in Medicine lecture at the Annual Meeting of the Royal College of Physicians and Surgeons of Canada, Halifax, Sept. 27, 1996.

(Original manuscript submitted Jan. 24, 1997; received in revised form Feb. 6, 1997; accepted Feb. 10, 1997)

Reprint requests to: Dr. Catherine P. M. Hayward, McMaster University Health Sciences Centre, Rm. 2N32, 1200 Main St. W, Hamilton ON L8N 3Z5; fax 905 521-2338

Contents

Abstract

In studies conducted about 8 years ago, the author and her colleagues raised a monoclonal antibody that recognized an uncharacterized human platelet protein with a reduced molecular mass of 155 kDa. Investigations of this protein's nonreduced structure yielded surprising findings: in its native state, it exists as massive disulfide-linked multimers millions of daltons in size, making it one of the largest proteins found in the human body. This feature led the author to designate this protein "multimerin." Multimerin is found in endothelial cells as well as in platelets. It originates from a single subunit protein, promultimerin, that undergoes extensive N-glycosylation, proteolytic processing and polymerization during biosynthesis. Recent data from the cloning and sequencing of its complementary DNA indicate that multimerin is a unique protein. Like von Willebrand factor, it has a massive repeating structure, but these proteins are unrelated. Multimerin's sequence contains the adhesive motif Arg-Gly-Asp-Ser, central coiled-coil sequences, several epidermal growth- factor-like motifs, and a globular domain that is similar to a protein-binding domain found in complement C1q and in collagens type VIII and X. Investigations of multimerin's function indicate that it binds the coagulation protein factor V and its activated form, factor Va. In platelets, but not in plasma, all of the biologically active factor V is complexed with multimerin. Multimerin may also have functions as an extracellular matrix or adhesive protein. Recently, members of 2 Canadian families with puzzling autosomal-dominant bleeding disorders were found to have a deficiency of platelet multimerin. Studies of these patients may provide a unique opportunity to evaluate the functions of multimerin.

Résumé

Au cours d'études réalisées il y a environ 8 ans, l'auteur et ses collègues ont produit un anticorps monoclonal qui reconnaissait une protéine de plaquette humaine non définie dont la masse moléculaire réduite était de 155 kDa. Des analyses de la structure non réduite de cette protéine ont donné des résultats étonnants : dans son état naturel, elle existe sous forme de multimères massifs à liaisons bisulfure dont la taille atteint des millions de daltons, ce qui en fait une des plus grosses protéines que l'on trouve dans le corps humain. Cette caractéristique a incité l'auteur à appeler cette protéine «multimérine». On trouve la multimérine dans les cellules endothéliales, ainsi que dans les plaquettes. Elle provient d'une seule protéine oligomère, la promultimérine, qui subit une N-glucosylation poussée, une transformation protéolytique et une polymérisation au cours de la biosynthèse. Des données récentes tirées du clonage et du séquençage de son ADN complémentaire indiquent que la multimérine est une protéine unique. Comme le facteur de von Willebrand, elle a une structure massive répétitive, mais les protéines n'ont aucuns liens entre elles. La séquence de multimérine contient le motif adhésif Arg-Gly-Asp-Ser, des séquences de spirales spiralées centrales, plusieurs motifs semblables au facteur de croissance épidermique et un domaine globulaire semblable à un domaine qui a un pouvoir de liaison avec les protéines que l'on trouve dans le C1q complémentaire et dans les collagènes de types VIII et X. Des analyses de la fonction de la multimérine indiquent qu'elle fixe le facteur V de la protéine de la coagulation et sa forme activée, le facteur Va. Dans les plaquettes, mais non dans le plasma, tout le facteur V actif sur le plan biologique forme un complexe avec la multimérine. La multimérine peut aussi agir comme matrice extracellulaire ou protéine adhésive. Récemment, on a trouvé chez des membres de 2 familles canadiennes atteints de troubles de saignement autosomiques dominants intrigants une carence de multimérine plaquettaire. Des études effectuées sur ces patients pourraient présenter une occasion unique d'évaluer les fonctions de multimérine.

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Introduction

 About 8 years ago, while determining the specificity of a monoclonal antibody raised against human platelets, I identified a previously uncharacterized platelet protein. My studies indicated that this protein had a reduced mobility of 155 kDa; however, investigations of its nonreduced structure yielded surprising findings.1,2 In its native state, this protein exists as massive disulfide-linked multimers (or polymers) that vary from 400 000 to many millions of daltons, making it one of the largest proteins in the human body.2 This feature led me to designate the native protein as multimerin.2 Although multimerin was initially identified in platelets, it has since been found in endothelial cells as well.3

This review describes the work that led to the discovery of multimerin, as well as subsequent studies that have characterized its structure, cells of origin, biosynthesis and function.1­7 It also reviews recent insights into multimerin's structure and function that have been attained through cloning and sequencing its complementary DNA.3 Finally, it looks at a puzzling bleeding disorder in several families in whom a deficiency of platelet multimerin has been found.8,9

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Background

An understanding of the function of a protein often comes from investigations of its structure. The multivalent structure of some proteins has an important influence on their biologic activity. For example, von Willebrand factor has a massive repeating structure that is critical for its function: the largest von Willebrand factor molecules (containing the highest number of ligand-binding sites) preferentially bind to platelets, and patients lacking the high-molecular-weight forms of von Willebrand factor have bleeding disorders.10­12

Many proteins exist as dimers or oligomers, but massive disulfide-linked polymeric proteins with repeating structures are rare. For years, the massive repeating structure of von Willebrand factor was thought to be unique, but the studies described in this report have shown that multimerin also is composed of massive, variably sized, disulfide-linked polymers (Fig. 1). However, multimerin and von Willebrand factor have different reduced and nonreduced mobilities (Fig. 1).1,2 The extremely large nonreduced size of multimerin is the likely reason that this protein was not recognized until recently, as its resolution requires gels that are capable of sieving a protein millions of daltons in size.1,2,5,6

As with von Willebrand factor, the repeating structure of multimerin is important for its function, with the largest polymers binding preferentially to the surface of activated platelets.2,5,6 Investigations comparing multimerin with other platelet proteins indicate that multimerin and von Willebrand factor are the 2 largest proteins -- and indeed the only massive, variably sized, disulfide-linked polymeric proteins -- present in platelets.1,2,5,6 Many similarities exist between multimerin and von Willebrand factor, but data derived from the recent cloning and sequencing of multimerin cDNA indicate that they are distinctly different.3

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Tissue and cellular distribution of multimerin

The hydrophilic properties of multimerin, its primary sequence and its secretion by cultured megakaryocytes are all consistent with a soluble protein.1­6 However, it is not present at detectable levels in the plasma.1,2,5,6 In platelets, it is sequestered within storage organelles, for regulated secretion in response to platelet activation.2,4 I later found it to be expressed by endothelial cells as well.3 Recent studies confirm that it is also stored in secretory granules in endothelial cells, for regulated secretion when these cells are exposed to agonists such as thrombin (unpublished data).

The expression of multimerin on the platelet surface can be used to study the process of platelet activation, as platelet activation leads to multimerin secretion and its subsequent binding to the external platelet membrane.1,2,4­6 Quantitative direct-binding studies indicate that the average number of multimerin monoclonal antibody-binding sites on a platelet increases from 600 to 4100 after activation by thrombin (Fig. 2).1,5,6 The membrane receptor that allows multimerin to bind to the platelet surface is not yet known but is under investigation. Comparison studies of multimerin and P-selectin expression on platelets indicate that antibodies to both proteins can be used as markers of platelet activation.1

After platelet activation by strong agonists such as thrombin, only small quantities of small multimerin oligomers are released from the platelets (Fig. 1); most of the secreted multimerin remains associated with the activated platelets.2,5,6 This may explain why multimerin is not measurable in plasma.

The distribution of multimerin within platelets is similar to that of von Willebrand factor.13,14 It is stored in an unusual, eccentric position within platelet *-granules.4,5,7,9 Double-labelling studies have indicated that multimerin colocalizes with von Willebrand factor within the electron-lucent zone of the alpha-granule, a region that has a similar ultrastructure to endothelial cell Weibel-Palade bodies.13 During platelet activation, there is secretion of the soluble proteins stored in platelet alpha-granules, and this moves multimerin out into the surface-connected cannalicular system.1,4­7 Some of the released multimerin then binds to the external platelet membrane.1,4­7

The sequestration of multimerin in intracellular stores and its secretion with granule release suggest that it plays a role in the response to vascular injury. Its storage within cells and its absence in plasma may aid in restricting and localizing its biologic functions.

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Biosynthesis of multimerin

Studies of the biosynthesis of multimerin have provided important insights into its structure, glycosylation and subunit composition. I have found only 2 cell types that synthesize multimerin: megakaryocytes and endothelial cells.3­6 A summary of the steps involved in multimerin biosynthesis is given in Fig. 3.

My initial studies of multimerin indicated that it is composed of multiple copies of a single building block, linked together by interchain disulfide bonds, forming 400-kDa trimers and larger polymers.1,2,5,6 The large polymers differ from the small oligomers only in the number of subunits that they contain.1,2,5,6

Although multimerin polymers are assembled from promultimerin subunits (Mr, reduced, 196 kDa), the protein undergoes proteolytic processing to create the smaller multimerin subunits found in the mature protein.4 When multimerin is synthesized by cultured cells, it contains large amounts of promultimerin.4 In contrast, the multimerin found in platelets is mainly composed of 155-kDa subunits and smaller quantities of a 170-kDa subunit. Some platelet lysates also contain traces of promultimerin.1,2,4­6 I was unable to obtain the N-terminal sequence from platelet multimerin because the protein was blocked, so the sites of proteolytic cleavage in promultimerin that produce the mature subunits are not yet known.3

Multimerin undergoes extensive N-linked glycosylation during biosynthesis.4 The mature protein is rich in complex forms of N-linked carbohydrates, which account for approximately one-third of its apparent molecular mass.4 In addition to glycosylation and proteolytic processing of the multimerin subunits, interchain disulfide bonds form during biosynthesis to link multiple copies of the subunits together.1,2,4

In contrast to von Willebrand factor, which is assembled from dimers,10 the smallest multimerin oligomer is a trimer.2 The mobility of its multimers (relative to those of von Willebrand factor) in denaturing gels suggest that multimerin may be assembled as multiples of trimers. However, multimerin's tertiary structure has not yet been characterized. The propolypeptide sequence of von Willebrand factor is thought to be important for its targeting and storage in organelles,10,15 but there is no homology between promultimerin and either von Willebrand factor or its propolypeptide.3 At present, the factors that target multimerin to regulated secretory granules during biosynthesis are unknown.

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The primary structure of multimerin

Recently, I completed the cloning and sequencing of the multimerin cDNA.3 The full-length cDNA encodes prepromultimerin, a protein 1228 amino acids in length with a 19-amino-acid signal peptide. Minus its signal peptide, the predicted protein size is 136 kDa, which is in close agreement with the apparent molecular mass of 132 kDa for deglycosylated promultimerin.3,4 Similar to the characteristics of the multimerin protein, the cDNA encodes a hydrophilic protein that contains 23 potential N-glycosylation sites and 19 cysteine residues that may be involved in both intra- and interchain disulfide bond formation.3 The sites of proteolytic cleavage that produce the mature subunits are not yet known.

Analyses of the deduced promultimerin sequence identified a number of sites that may be important for structure and function (Fig. 4).3 Promultimerin contains an RGDS (Arg-Gly-Asp-Ser) domain, an amino acid sequence that is present in many proteins with adhesive functions.16 This finding suggests a possible role for multimerin as an adhesive molecule.3 Promultimerin also contains both complete and partial epidermal growth factor (EGF)-like domains, which are structural motifs present in proteins with diverse functions.3,17 The consensus sequence for EGF-like domains contains paired cysteine residues that form intrachain disulfide bonds.17 The partial EGF-like domain in promultimerin lacks the first cysteine residue in EGF-like domains.3 The functions of EGF-like domains include protein binding;17 however, the roles of these domains in promultimerin are not yet known. Although the proteins rich in EGF-like domains are the proteins that have the highest homology scores with promultimerin, this similarity is restricted to the regions of promultimerin containing the complete EGF-like and partial EGF-like domains.3

Homology analyses indicate that several other regions of promultimerin also have similarities to other proteins.3 The central domain of promultimerin resembles the rod-like tail of myosin and other coiled-coil proteins, and contains sequences that are predictive of coiled-coil structures.3 This domain may be important for subunit interactions and tertiary structure.

Comparisons of promultimerin with known proteins also indicate similarities between its carboxyl-terminal domain and a trimeric, globular head structural motif found in several other proteins, including complement C1q and collagens type VIII and X.3,18­26 In complement C1q, the globular head domain is the site where the protein binds to IgG and other complement activator molecules.26 However, in collagens type VIII and X, the globular head domains have a different function, and interact with each other to form mesh-like structures in the extracellular matrix.27,28 On the basis of our knowledge of homologous proteins, the globular head domain in promultimerin is a likely site for protein interactions, but whether it functions in homotypic or heterotypic protein interactions is not yet known.

Since the homology observed between promultimerin and other proteins is restricted to specific domains, promultimerin does not belong to any known protein family.3 I had anticipated that there might be similarities between multimerin and von Willebrand factor, based on their similar protein trafficking and storage, but comparisons of their amino acid sequences indicate that there is no significant homology between them.3,29

Northern analyses indicate that the prepromultimerin messenger RNA is present in highly vascular tissues, in megakaryocyte cell lines and in platelets, which is consistent with the distribution of the multimerin protein in endothelial cells, megakaryocytes and platelets.3,4 Although the cDNA sequence for prepromultimerin was determined using endothelial cell libraries,3 analyses of prepromultimerin mRNA (using reverse transcription and polymerase chain reaction amplification) indicate that an identical transcript is expressed in megakaryocytes, megakaryocytic cell lines and endothelial cells (Fig. 5).

The importance of the various domains identified in promultimerin for structure and function and the factors that regulate its tissue-specific expression are not yet known. Based on knowledge derived from homologous proteins, I postulate that the RGDS, globular head and EGF-like domains will prove to be important for multimerin function and for its interactions with other proteins.

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Interaction of multimerin with coagulation factor V

Factor V is a key regulatory cofactor in coagulation that is found both within platelets and in plasma.30,31 During coagulation, factor V is converted to factor Va, forming part of the prothrombinase complex and functioning to accelerate thrombin generation and clot formation.30,32 Collaborative studies of platelet proteins that might regulate factor V activity led us to identify multimerin as the major protein that binds factor V in platelets.7

Multimerin specifically binds both factor V and its activated form, factor Va. This interaction is mediated by the light-chain domain of factor Va.7 All of the biologically active factor V stored in platelets is complexed with multimerin; multimerin immunodepletion results in a loss of all detectable factor V activity in platelets.7 Within platelet alpha-granules, factor V is found together with multimerin, confirming the presence of factor V-multimerin complexes in intact platelets (Fig. 6).7 Investigations using purified proteins indicate that these complexes can form in either the presence or absence of other platelet proteins.7

Although factor V is bound to multimerin in resting platelets, this is not the case after thrombin activation:7 most of the factor Va bound to the membrane of activated platelets is not associated with multimerin. The mechanism for this dissociation is not yet understood, since when purified multimerin-factor V complexes are treated with thrombin, the factor Va generated remains bound to multimerin.7 The exposure of higher affinity factor V binding sites on platelets could be one of the factors that contributes to the dissociation of multimerin and factor Va during platelet activation. An interesting and similar analogy of a coagulation cofactor binding to a massive multimeric protein exists for factor VIII and von Willebrand factor.33 This interaction is thought to protect factor VIII from degradation.

Many large proteins have more than one function. The association of multimerin and factor V in platelets suggests that multimerin may function as a factor V carrier protein, but recent studies in my laboratory suggest that it may also play a role as an extracellular matrix protein (unpublished data).

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Multimerin deficiency in an inherited bleeding disorder

Knowledge of clinical disorders associated with deficient proteins has provided major insights into the function of many proteins. In this case, the interaction between multimerin and factor V in platelets led me to investigate patients with factor V deficiency states for possible multimerin abnormalities.

Most patients with inherited factor V deficiencies are deficient in both platelet and plasma factor V, as factor V is expressed in both megakaryocytes and hepatocytes.30 However, about 13 years ago a bleeding disorder (originally designated as factor V Quebec), affecting primarily the platelet stores of factor V, was reported.34 Platelets from patients with factor V Quebec have reduced factor V function, but they support coagulation normally when exogenous factor V is supplied. Our studies showing that factor V was bound to multimerin in platelets led me to investigate these patients. Specifically, I wondered if the fact that their platelet factor V was abnormal could be explained by a defect or deficiency in multimerin.

Indeed, I found the multimerin antigen levels in these patients to be markedly reduced: their levels range from 5% to 27% of the normal platelet pool multimerin content, compared with healthy controls, whose levels range from 45% to 214% (Fig. 7).8 The Quebec patients have a serious hemostatic defect characterized by mucocutaneous bleeding episodes and moderate to severe hemorrhage after surgery or trauma (Table 1). Several individuals have suffered spontaneous intracranial hemorrhage, and deaths due to bleeding have occurred in several family members. In contrast to most bleeding disorders, this defect is inherited as an autosomal-dominant trait (Fig. 8).8,34 Although the multimerin levels in these patients are extremely low, their multimerin subunits and multimers are normal.8 Their platelet factor V has both quantitative and qualitative abnormalities, which may be due to abnormal proteolytic degradation.8,34 However, these patients are not deficient in all alpha-granular proteins,8 as occurs in gray platelet syndrome.35­38

Although I first studied these patients to determine whether they had abnormalities in multimerin, I also found multiple abnormalities in the profile of their platelet lysate and releasate proteins, when separated on denaturing gels.8,9 In addition, many of their alpha-granular proteins are degraded.8,9,39The proteins that are known to be degraded include factor V, von Willebrand factor, thrombospondin, fibrinogen, fibronectin, osteonectin and P-selectin, which are all stored in platelet *-granules.8,9,39 However, we found no abnormalities in their platelet external membrane glycoproteins, cytosolic calpain, or CD63 (a dense and lysosomal granular membrane protein).9 Although many alpha-granular proteins are degraded in this disorder, they are still found in their normal intracellular location.9 Together, these findings suggest that the pathologic protein degradation in this disease may be restricted in its intracellular location so that only the alpha-granular proteins are altered. Because of the many abnormalities in the platelets, we decided to redesignate this disorder "the Quebec platelet disorder."9

Recently we identified a second family, also from Quebec, with this disorder.9 Similar to the patients in the first family, these individuals have a quantitative platelet-multimerin deficiency (with levels ranging from less than 5% to 9% of the normal platelet pool).8,9 Many of their alpha-granular proteins are degraded, and they comigrate with the degradation products found in patients from the other family.8,9 These findings suggest that the same protease (or proteases) may be degrading the platelet proteins in these 2 sets of patients. However, we have not found any links in the ancestry of the two families.9 Whether they share a common early ancestor or whether they suffer from similar but distinct genetic mutations is not yet known.

Another curious and unexplained feature of the disorder, seen in both families, is that many patients are thrombocytopenic, with platelet counts that range from 80 to 200×109/L. 8,9 In addition, they have defective epinephrine aggregation, despite normal platelet alpha2-adrenergic receptors.8,9

Curiously, no blood products (including platelet concentrates) have been effective in controlling the hemorrhagic episodes in these patients.8,9 This finding suggests that factors other than replacing deficient or abnormal platelet proteins have a major influence on the bleeding. The release of abnormal alpha-granular proteins during blood clotting may interfere with normal hemostasis and contribute to the bleeding.

The explanation for the multimerin deficiency and its contribution to bleeding in the Quebec platelet disorder are not yet known. In contrast to the degradation we demonstrated in many other alpha-granular proteins, we found only quantitative abnormalities in multimerin.8,9 Although we had originally investigated these patients for a defect in multimerin, their abnormalities suggest that this disorder could be due to a defective protease, a deficiency of a protease inhibitor, or a protease in an abnormal location.

Most of the characterized platelet disorders affect a single protein, but the Quebec platelet disorder, similar to gray platelet syndrome, is associated with multiple glycoprotein abnormalities.35­38Other patients with undefined hemostatic disorders may also suffer from this form of platelet disorder. In some circumstances, analyses of platelet glycoproteins can be helpful in evaluating bleeding problems. Analyses of platelet multimerin can be used for diagnostic purposes, as my studies of unrelated individuals with a variety of bleeding disorders indicate that the multimerin abnormalities are specific to the Quebec platelet disorder.8,9

These studies have identified the first clinical disorder associated with multimerin deficiency.

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Conclusion

Since the discovery of multimerin in 1990, much knowledge has been acquired about its structure, tissue distribution, biosynthesis and function. The recent cloning and sequencing of the multimerin cDNA has confirmed its unique identity, provided clues to possible new functions and laid the groundwork for future structure and function investigations. Multimerin may serve as a carrier protein for the factor V stored in platelets, and it likely has other important functions as well, perhaps as an extracellular matrix or adhesive protein. Studies of several Canadian families with inherited multimerin deficiency and degradation of platelet alpha-granular proteins have led to the discovery of a novel platelet disorder and have provided new diagnostic tools for investigating patients with undefined bleeding disorders. Studies of these patients may provide a unique opportunity to evaluate the functions of multimerin and other proteins secreted by platelets.

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Acknowledgements

I am grateful to my former PhD supervisor, Dr. John G. Kelton, to the individuals in his laboratory, and to Drs. John A. Hassell, Richard A. Rachubinski and Peter Horsewood for their guidance and mentorship during my doctoral work. I would also like to gratefully acknowledge the contributions to the studies cited of my collaborators, Drs. Georges E. Rivard, Elisabeth M. Cramer, William H. Kane, Graham Côté, Michael Nesheim, Dorothy F. Bainton, Ron H. Stead, Jeanne Drouin, and Thomas Podor, and the individuals in their laboratories. These studies were supported by operating grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario. Dr. Hayward is a Research Scholar of the Heart and Stroke Foundation of Ontario and the recipient of an Ortho Biotech/ASH Scholar Award.

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