The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)

Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Modified clotting properties of fibrinogen in the presence of acetylsalicylic acid in a purified system.

To assess how treatment with acetylsalicylic acid (ASA) alters the fibrin network structure, clotting was initiated in purified fibrinogen incubated with ASA by adding thrombin. Clotting time and maximum absorbance of the fibrin aggregation curve were used to demonstrate the potential of fibrin generation. The results showed that the clotting properties of fibrinogen decreased and that the affinity of plasminogen to fibrin or thrombin inhibition by antithrombin increased if plasminogen or antithrombin, respectively, were present in the reaction system. The effect of ASA varied in a dose dependent manner. It was concluded that ASA may directly or indirectly confer positive or negative effects on the stability of the fibrin clot and that the balance between these effects may be regulated by the ASA dose.

Fibrinogen non-inherited heterogeneity and its relationship to function in health and disease.

In healthy individuals fibrinogen occurs in more than one million non-identical forms because of the many possible combinations of biosynthetically or postbiosynthetically modified or genetically polymorphic sites. The various forms may show considerable differences in their functional properties. Normal variant sites are due to alternative splicing, modification of certain amino acid residues, and proteolysis. Both the A alpha and the gamma chain occur in two splice forms, and it is known that only the shorter gamma chain can interact with platelets, but the longer may bind thrombin and factor XIII. Many types of posttranslationally modified amino acid residues are present in fibrinogen. The A alpha chain is partially phosphorylated at two sites, possibly leading to protection against proteolysis. The B beta chain is N-glycosylated and partially proline hydroxylated, each at one site. The gamma chain is N-glycosylated at one site and the longer splice form doubly tyrosine-sulfated. The glycosylations are believed to protect against polymerization and proteolysis. All three chains are partially oxidized at methionine residues and deamidated at asparagine and glutamine residues. The A alpha and gamma chain are partially carboxy-terminally degraded by proteolysis, the shorter forms causing a decrease in polymerization, crosslinking, and clot stability. Abnormal variants occur in patients with diabetes mellitus, in the form of glycated lysine residues; in patients with certain types of cancer, in the form of crosslinked degradation products; in patients with certain types of autoimmune disease, in the form of complexes with antibodies; in cigarette smokers; and in individuals treated with acetylsalicylic acid, in the form of acetylated lysine residues.

Antifibrinogen IgG, fibrinogen, and Clq complexes circulating in a hypodysfibrinogenemic proband. Isolation, stoichiometry, and partial characterization.

Circulating antifibrinogen antibodies have been reported in rare afibrinogenemic propositi, apparently occurring following fibrinogen replacement therapy, but immune complexes have not been described. In this report we describe circulating immune complexes formed by a monoclonal antifibrinogen IgG in a heterozygous hypodysfibrinogenemic (A alpha 16 Arg-->Cys) proband. Estimated by partial protein sequence and by other analyses, each immune complex consisted of one fibrin(ogen), one C1q, and 3-4 IgG molecules. The complexes were cryoprecipitable, a property also displayed by mixtures of proband IgG and normal fibrinogen. Indicating that both D and E domains were necessary for this behavior, cryoprecipitability was abolished by preincubation of the isolated IgG with either isolated normal fibrinogen fragment D100 or E. Consistent with the crossreactivity of the IgG with normal and mutant fibrinogen, the results suggest that the primary epitope resides on a D-E locus on the fibrin polymer formed by normal and abnormal molecules containing the uncleaved (or mutant) peptide A.

Methylation of histone H3 by coactivator-associated arginine methyltransferase 1.

The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177]. To further evaluate the significance of H3 methylation, we studied the kinetics and site specificity of its modification by CARM1. Affinity-purified CARM1 methylated recombinant chick H3, which is free of posttranslational modifications, and calf thymus H3, which is heterogeneous with regard to preexisting modifications, equally well, exhibiting a V(max) of 4500 pmol min(-1) (mg of enzyme)(-1) and an apparent K(m) for H3 of < or = 0.2 microM. The catalytic efficiency (k(cat)/K(m)) of CARM1 toward H3 was at least 1000 times that toward R1 (GGFGGRGGFGG-amide), a highly effective substrate for protein arginine methyltransferase 1. Peptide mapping of 3H-methyl-labeled H3 indicated methylation at Arg-2, Arg-17, and Arg-26 in the N-terminal region and at one or more of four arginines (128/129/131/134) at the C-terminus. Two of the N-terminal sites, Arg-17 and Arg-26, occur in the sequence KAXRK and appear to be more efficiently methylated than Arg-2. CARM1 catalyzed formation of N(G),N(G)-dimethylarginine (asymmetric) but little or no N(G),N'(G)-dimethylarginine (symmetric) and no form of methyllysine. Amino acid analysis of untreated calf thymus H3 revealed that 3.7% of the molecules naturally contain asymmetric dimethylarginine and/or monomethylarginine. Our findings support the hypothesis that methylation of H3 may be involved in the mechanism of transcriptional coactivation by CARM1 of genes whose expression is under the control of nuclear receptors.

The crystal structure of modified bovine fibrinogen.

Here we report the crystal structure at approximately 4-A resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments. We have visualized the entire coiled-coil region of the molecule, which has a planar sigmoidal shape. The primary polymerization receptor pockets at the ends of the molecule face the same way throughout the end-to-end bonded filaments, and based on this conformation, we have developed an improved model of the two-stranded protofibril that is the basic building block in fibrin. Near the middle of the coiled-coil region, the plasmin-sensitive segment is a hinge about which the molecule adopts different conformations. This segment also includes the boundary between the three- and four-stranded portions of the coiled coil, indicating the location on the backbone that anchors the extended flexible Aalpha arm. We suggest that a flexible branch point in the molecule may help accommodate variability in the structure of the fibrin clot.

Digestion of C1q collagen-like domain with MMPs-1,-2,-3, and -9 further defines the sequence involved in the stimulation of neutrophil superoxide production.

C1q, a subunit of the first component (C1) of the classical complement pathway, binds to neutrophils via its collagen-like region (C1q-CLR) stimulating superoxide production. We previously identified a region of C1q-CLR, defined by fragments generated by trypsin and endoLys-C digestion, that was required for triggering this respiratory burst. To further localize that critical site, purified human C1q was digested with pepsin to generate C1q-CLR, and subsequently cleaved with the matrix metalloproteinases, MMP-1, MMP-2, MMP-3, and MMP-9. Digestion of C1q-CLR with any of these MMPs did not alter the circular dichroism spectra, demonstrating that the fragments generated had maintained the secondary structure observed in the native molecule. All fragments retained the ability to trigger superoxide production by neutrophils. Analysis of the amino acid sequences of the purified cleavage products (none of which are identical to the published cleavage site specificities for these enzymes) demonstrated that it is the C-chain, but not the A-chain of C1q, that is critical for stimulating this activity, and thus may be a target for future therapeutic intervention.

Crotalase, a fibrinogen-clotting snake venom enzyme: primary structure and evidence for a fibrinogen recognition exosite different from thrombin.

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence [see text] characteristic of a serine proteinase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin's fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin's YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme's total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm(-1) is 15.2.

On the identification of beneficial and detrimental molecular forms of fibrinogen.

Fibrinogen is a central protein in blood coagulation. A functioning circulation system requires a precise balance between fibrin formation and removal, i.e. between the interaction of fibrin(ogen) with thrombogenic and fibrinolytic components of the blood. Fibrinogen and fibrin have also significant roles in wound healing, in tumor growth and metastasis as well as in defense mechanisms. All functions and interactions are mediated by specific structural elements of the molecule. Already in healthy individuals fibrinogen occurs in over a million nonidentical forms due to posttranslational modifications and genetic polymorphism. The various forms may show considerable differences in their functional properties. Alterations in distributions among preexisting forms as well as additional forms have been observed to accompany many types of disease. Furthermore, certain forms have been correlated with an increased risk to acquire disease. Monitoring the levels of various molecular forms is expected to be of considerable diagnostic and prognostic value in many types of disease.

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

Structural examination of the influence of phosphorylation on the binding of fibrinopeptide A to bovine thrombin.

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.

cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro.

The complement protein C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) are structurally similar molecules that enhance phagocytic function in vitro. Monoclonal antibodies R3 and R139, which inhibit the enhancement triggered by these three ligands, were used to purify a 126,000 M(r) cell surface protein designated C1qR(P). Amino acid sequence was obtained and the corresponding cDNA was cloned. C1qR(P) is a novel type I membrane protein with the following putative structural elements: a C-type carbohydrate recognition domain, five EGF-like domains, a transmembrane domain, and a short cytoplasmic tail. All peptides identified by amino acid sequencing are encoded by the cDNA. Additionally, an anti-peptide antiserum was generated, which is reactive with C1qR(P). The data indicate that the cloned cDNA encodes the receptor that plays a role in C1q/MBL/SPA-mediated removal or destruction of pathogens and immune complexes by phagocytosis.

Photo-oxidation of histidine as a probe for aminoterminal conformational changes during fibrinogen-fibrin conversion.

Fibrinogen is known to become unclottable when irradiated with light in the presence of methylene blue, the loss of clottability being due to photo-oxidation of the histidine at position 16 of the B beta chain. In the present investigation it could be demonstrated that not only this histidine but also the one at position 24 of the A alpha chain was modified and that the rates of modification could be modulated by fibrinopeptide release, polymerization inhibition and denaturation. Accordingly, the histidine modifications can be used as probes for surface accessibility of and conformational differences among the various forms of the protein. Both histidines are shielded by the fibrin polymer structure. Fibrinopeptide A release alone leads to maximal protection of the one in the A alpha chain, but only partial protection of the one in the B beta chain. Subsequent fibrinopeptide B release leads to maximal protection of the one in the B beta chain. The differential effects indicate that two conformational changes have occurred. Polymerization inhibition reverses the protective effect. Denaturation leads to maximal and equal modification in all samples as a consequence of the loss of native conformation.

Localization of the site on the complement component C1q required for the stimulation of neutrophil superoxide production.

C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C. The resulting fragments were separated by gel filtration chromatography and analyzed functionally (activation of the respiratory burst in neutrophils) and structurally. Cleavage of C1q-CLR with endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with trypsin under conditions permitting optimal cleavage, the ability of C1q-CLR to stimulate superoxide production in neutrophils was completely abrogated. Fractionation of the digests obtained with the two enzymes and identification by amino acid sequencing permitted localization of the receptor interaction site to a specific region of the C1q-CLR. Circular dichroism analyses demonstrated that cleavage by trypsin does not denature the remaining uncleaved collagen-like structure, suggesting that after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat denaturation of C1q-CLR also abrogated all activity. Thus, a specific conformation conferred by the collagen triple helix constitutes the functional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this response.