Johan Stenflo

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions.

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.

Binding of Ca2+ to normal and dicoumarol-induced prothrombin

Abstract The Ca 2+ binding properties of normal bovine prothrombin have been studied and compared with those of an abnormal bovine prothrombin induced by dicoumarol. The normal prothrombin binds up to 10–12 Ca 2+ per mole of protein. The three first Ca 2+ were bound to sites which exhibited positive cooperativity. A Ca 2+ dependent conformational change was demonstrated during the binding of the first three Ca 2+ . In contrast with normal prothrombin, the dicoumarol-induced prothrombin had only one high affinity binding site. No ligand-induced conformational change was detected in this prothrombin.

Post-translational modifications in proteins involved in blood coagulation.

Blood coagulation and its anticoagulant counterpart, the protein C (PC) system, proceed via the formation of cellassociatedmacromolecular enzyme complexes that interact in a precisely regulated manner. In the final step of coagulation thrombin is generated, which cleaves soluble fibrinogen to form the insoluble fibrin monomers that constitute the structural foundation of a blood clot [1,2]. Post-translational protein modifications play pivotal roles in both blood coagulation and the PC anticoagulant system. The relatively high concentration of most of the blood coagulation proteins enabled many of them to be purified to homogeneity some 30–40 or more years ago. Advanced chemical characterization and protein sequencing became possible, which in time resulted in the identification of several post-translational amino acid modifications. These developments, and the interest in blood coagulation and coagulation disorders, led to the identification of three types of modified amino acids in coagulation proteins before they were found in proteins belonging to other systems. The first was c-carboxyglutamic acid (Gla) in 1974, which is formed by vitamin K-dependent c-carboxylation of glutamic acid residues [3–5]. Then followed erythro-b-hydroxyaspartic acid (Hya) and erythro-b-hydroxyasparagine (Hyn), which are formed by hydroxylation of aspartic acid and asparagine residues, respectively [6,7]. At that time, blood coagulation was described by the so-called cascade scheme [2,8,9]. It called attention to the signal amplification that is required to obtain the amounts of thrombin necessary to convert soluble fibrinogen to insoluble fibrin and it illustrated the role of enzymatically active macromolecular complexes. Moreover, the importance of binding to cell surfaces, i.e. that the coagulation process to a large extent is an example of chemistry in two (rather than three) dimensions was only beginning to emerge. The identification of Gla, stimulated by a desire to learn about the mode of action of warfarin [10], indirectly led to the purification of PC, and a few years later protein S and thrombomodulin [11–15]. With these proteins identified, and with the novel co-factor role of factor V established as a result of research on resistance to activated protein C (APC), most of the key components of the PC anticoagulant system had been identified [16]. Lately, the coagulation system has been refined because of new insights into the roles of the various cell types involved [17]. Hence, the initiation phase of coagulation is now known to commence on tissue factor (TF)-bearing cells such as macrophages, followed by an amplification phase involving platelet activation and activation of FV and FVIII. In the third phase, the propagation phase, bulk amounts of thrombin are generated on the surface of newly activated platelets [17]. The activity of the serine proteases generated during this sequence of reactions is under rigorous control, mediated by the TF pathway inhibitor and antithrombin; the latter a so-called serpin [18]. Likewise, APC and its co-factors, protein S and FV, regulate the activity of the activated forms of two homologous co-factors, FVIIIa and FVa [19]. In this review, we describe the post-translational modifications that occur in blood coagulation proteins and, where known, their functional implications. In the integrated defense system there are no borders between blood coagulation, the complement system, and the immune system. We have, however, taken a conservative approach and will deal only with those modifications that are found in the traditional coagulation factors, including the PC anticoagulant system and antithrombin. Seven years ago an excellent review of the same field was published [20].

Crystal structure of protein C inhibitor provides insights into hormone binding and heparin activation.

Protein C inhibitor (PCI) is a member of the serpin family that has many biological functions. In blood it acts as a procoagulant, and, in the seminal vesicles, it is required for spermatogenesis. The activity of PCI is affected by heparin binding in a manner unique among the heparin binding serpins, and, in addition, PCI binds hydrophobic hormones with apparent specificity for retinoids. Here we present the 2.4 A crystallographic structure of reactive center loop (RCL) cleaved PCI. A striking feature of the structure is a two-turn N-terminal shortening of helix A, which creates a large hydrophobic pocket that docking studies indicate to be the retinoid binding site. On the basis of surface electrostatic properties, a novel mechanism for heparin activation is proposed.

Explorative investigation of biomarkers of brain damage and coagulation system activation in clinical stroke differentiation

IntroductionA simple and accurate method of differentiating ischemic stroke and intracerebral hemorrhage (ICH) is potentially useful to facilitate acute therapeutic management. Blood measurements of biomarkers of brain damage and activation of the coagulation system may potentially serve as novel diagnostic tools for stroke subtypes.MethodsNinety-seven stroke patients were prospectively investigated in a multicenter design with blood levels of brain biomarkers S100B, neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) as well as a coagulation biomarker, activated protein C – protein C inhibitor complex (APC-PCI), within 24 hours of symptom onset.ResultsEighty-three patients (86 %) had ischemic stroke and fourteen patients (14 %) had ICH. There were no differences in S100B (p = 0.13) and NSE (p = 0.67) levels between patients with ischemic stroke or ICH. However, GFAP levels were significantly higher in ICH patients (p = 0.0057). APC-PCI levels were higher in larger ischemic strokes (p = 0.020). The combination of GFAP and APC-PCI levels, in patients with NIHSS score more than 3, had a sensitivity and negative predictive value of 100 % for ICH in our material (p = 0.0052).ConclusionThis exploratory study indicated that blood levels of biomarkers GFAP and APC-PCI, prior to neuroimaging, may rule out ICH in a mixed stroke population.

Protein S, a new vitamin K-dependent protein from bovine plasma

Five zymogens of serine amidases that require vitamin K for their biosynthesis have been purified from bovine plasma and chemically characterized. Prothrombin (factor II), factor VII, factor IX and factor X are the four traditional vitamin K-dependent clotting factors [ 11, whereas the fifth protein, called protein C [2] was isolated and characterized only recently [3-61. Evidence has been presented [7] indicating that protein C has a regulatory function in blood coagulation. Protein C can be activated to a serine amidase by the factor X activator from Russel’s viper venom or by thrombin [3]. All these proteins contain the vitamin K-dependent y-carboxyglutamic acid residues. Another protein called protein S which occurs in very low concentration in human plasma was purified (81. In the course of our purification of protein C from large volumes of bovine plasma we have found a previously unknown vitamin K-dependent protein, and devised a simple method for its purification. A monospecific antiserum against this protein did not react with any of the other vitamin K-dependent plasma proteins. The amino terminal sequence of this protein is similar to that of human protein S. Our antiserum against this protein also reacts with a vitamin K-dependent protein purified [9] and considered to be the bovine counterpart to human protein S. Our procedure for the purification of bovine protein S and some of its properties is described here.

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods†

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.

Identification of γ-carboxyglutamic acid residues in bovine factors IX and X, and in a new vitamin K-dependent protein

The four vitamin K-dependent plasma proteins participating in blood coagulation (prothrombin, factors VII, IX and X) are zymogens of serine endopeptidases [ 1,2] . These proteins bind calcium ions and can be adsorbed to barium citrate and similar salts of divalent cations. Recently, a new fifth vitamin K-dependent protein, arbitrarily referred to as protein C, was isolated from bovine plasma [3] . Also, this protein binds calcium and can be activated to a serine esterase [4]. Its biological function is still unknown. The binding of calcium by these proteins is a result of vitamin K action, since prothrombin synthesized under the influence of vitamin K antagonists does not bind calcium [5,6]. Prothrombin, factors VII, and IX are single chain structures whereas factor X and protein C have two polypeptide chains [l-3] . The calcium binding vitamin K-dependent structures are in the NHz-terminal parts of the polypeptide chains and those for factor X and protein C, in the NHz-terminal parts of the light chains [l] . In these parts of the molecules, there is a strong amino acid sequence homology between the proteins. Recently a new amino acid, y-carboxyglutamic acid, was found in prothrombin [7-91. The amino acid is formed by vitamin K-dependent carboxylation of the first 10 glutamic acid residues in prothrombin [ 1 O-l 21. Identification of y-carboxyglutamic acid in sequence work is complicated by the fact that the amino acid decarboxylates when heated in the acid form. We have designed a method for mass spectrometric determination of y-carboxyglutamic acid as methylesterified thiohydantoin derivative [ 111. The method allows direct identification of y-carboxyglutamic acid residues during automated sequenator degradation of peptides and proteins. The complete amino acid sequences of prothrombin and factor X were recently published [13--l 51. In factor X, however, the exact positions of the y-carboxyglutamic acid residues have not yet been unambiguously established, although it has been shown that a 39-residue peptide from the vitamin K-dependent part of prothrombin has approximately one extra carboxyl group per glutamic acid residue [ 161 The light chains of factor X and protein C lend themselves well to direct sequenator degradation. We, therefore, used the mass spectrometric method to locate the y-carboxyglutamic acid residues in these proteins.

Structure of Native Protein C Inhibitor Provides Insight into Its Multiple Functions

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into β-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.

The High Affinity Calcium-binding Sites in the Epidermal Growth Factor Module Region of Vitamin K-dependent Protein S

Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; β-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; β-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5′-Br2BAPTA, we have now determined the Ca2+affinity of recombinant fragments containing EGF modules 1–3, 1–4, 2–3, and 2–4. EGF modules 1–4 and 2–4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10−10 to 10−8 m in the absence of salt and from 10−8 to 10−6 m in the presence of 0.15 m NaCl. In contrast, in EGF 1–3 and EGF 2–3, the Ca2+ affinity is 2–4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1–4 and EGF 2–4 is 103–105-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ligands. The structural basis for the difference in Ca2+affinity is not yet understood.