Dominic W. Chung

Crystal structure of a 30 kDa C-terminal fragment from the γ chain of human fibrinogen

Abstract Background: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, ( α β γ ) 2 , held together by 29 disulfide bonds. The globular C terminus of the γ chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. Results: The X-ray crystallographic structure of the 30 kDa globular C terminus of the γ chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. Conclusions: The polymerization domain in the γ chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13.

Elevated plasma von Willebrand factor (VWF) and low ADAMTS13 activity have been reported in several inflammatory states, including sepsis and acute respiratory distress syndrome. One hallmark of inflammation is neutrophil activation and production of reactive oxygen species, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). HOCl is produced from hydrogen peroxide and chloride ions through the action of myeloperoxidase. HOCl can oxidize methionine to methionine sulfoxide and tyrosine to chlorotyrosine. This is of interest because the ADAMTS13 cleavage site in VWF, the Tyr(1605)-Met(1606) peptide bond, contains both oxidation-prone residues. We hypothesized that HOCl would oxidize either or both of these residues and possibly inhibit ADAMTS13-mediated cleavage. We therefore treated ADAMTS13 substrates with HOCl and examined their oxidative modification by mass spectrometry. Met(1606) was oxidized to the sulfoxide in a concentration-dependent manner, with complete oxidation at 75muM HOCl, whereas only a miniscule percentage of Tyr(1605) was converted to chlorotyrosine. The oxidized substrates were cleaved much more slowly by ADAMTS13 than the nonoxidized substrates. A similar result was obtained with multimeric VWF. Taken together, these findings indicate that reactive oxygen species released by activated neutrophils have a prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13.

Nucleotide Sequences of the Three Genes Coding for Human Fibrinogen

Fibrinogen is synthesized in the liver by hepatic parenchymal cells and is secreted into the circulation (1). Hepatic synthesis of fibrinogen is constitutive but the rate can be modulated by a number of physiological and nonphysiological factors. The three chains of fibrinogen are encoded by distinct species of mRNA that are derived from the expression of three single copy genes (2, 3). Present evidence indicates that the three genes of human fibrinogen are linked and are located in a region that extends approximately 45 kb on chromosome 4q23-q32 (4). The genes are arranged in the order of γ-Aα-Bβ. The γ and Aα genes are transcribed in the same direction while the Bβ gene is transcribed in the opposite direction.

Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNAs. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNAs that code for platelet proteins.

Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS-13).

Summary.  Background: Over the last 4 years ADAMTS‐13 measurement underwent dramatic progress with newer and simpler methods. Aims: Blind evaluation of newer methods for their performance characteristics. Design: The literature was searched for new methods and the authors invited to join the evaluation. Participants were provided with a set of 60 coded frozen plasmas that were prepared centrally by dilutions of one ADAMTS‐13‐deficient plasma (arbitrarily set at 0%) into one normal‐pooled plasma (set at 100%). There were six different test plasmas ranging from 100% to 0%. Each plasma was tested ‘blind’ 10 times by each method and results expressed as percentage vs. the local and the common standard provided by the organizer. Results: There were eight functional and three antigen assays. Linearity of observed‐vs.‐expected ADAMTS‐13 levels assessed as r2 ranged from 0.931 to 0.998. Between‐run reproducibility expressed as the (mean) CV for repeated measurements was below 10% for three methods, 10–15% for five methods and up to 20% for the remaining three. F‐values (analysis of variance) calculated to assess the capacity to distinguish between ADAMTS‐13 levels (the higher the F‐value, the better the capacity) ranged from 3965 to 137. Between‐method variability (CV) amounted to 24.8% when calculated vs. the local and to 20.5% when calculated vs. the common standard. Comparative analysis showed that functional assays employing modified von Willebrand factor peptides as substrate for ADAMTS‐13 offer the best performance characteristics. Conclusions: New assays for ADAMTS‐13 have the potential to make the investigation/management of patients with thrombotic microangiopathies much easier than in the past.

The Role of βγ and αγ Complexes in the Assembly of Human Fibrinogen

The role of αγ and βγ dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing β and γ chains and generating βγ complexes were fused with cells expressing only the α chain. Likewise, when cells co-expressing α and γ chains and generating αγ complexes were fused with cells expressing only the β chain, fibrinogen was also formed and secreted. The relative amounts of αγ or βγ intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the β chain was limiting, the αγ dimer was the predominant intermediate; likewise, when the α chain was limiting, the βγ complex was the predominant intermediate. The incorporation of preformed αγ and βγ complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of αγ and βγ complexes as functional intermediates in the assembly of fibrinogen.

Shear stress–induced unfolding of VWF accelerates oxidation of key methionine residues in the A1A2A3 region

VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H(2)O(2), we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-of-function conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13.

Fibrin Clot Structure and Mechanics Associated with Specific Oxidation of Methionine Residues in Fibrinogen

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.

The Assembly of Human Fibrinogen THE ROLE OF THE AMINO-TERMINAL AND COILED-COIL REGIONS OF THE THREE CHAINS IN THE FORMATION OF THE αγ AND βγ HETERODIMERS AND αβγ HALF-MOLECULES

Fibrinogen is a plasma protein consisting of six polypeptide chains which are linked by disulfide bonds. During protein synthesis, assembly of the molecule proceeds through the formation of αγ and βγ heterodimers followed by the generation of αβγ half-molecules and dimerizing to generate the mature six-chain molecule. In the present study, sequences required for the formation of the αγ and βγ heterodimers were examined in stably transfected baby hamster kidney cells expressing combinations of normal as well as modified polypeptide chains. Deletion of the amino terminus and the proximal first half of the coiled-coil region of the three fibrinogen chains had little or no effect on heterodimer and half-molecule formation. These deletions, however, did prevent half-molecules from forming the six-chain molecule. Deletion of the distal second half of the coiled-coil region of each chain completely prevented the assembly process. Point mutations in the second half of the coiled-coil region also indicated that hydrophilic residues that form ion pairs between interacting chains were not critical in the formation of the heterodimeric complexes. These results suggest that the initial formation of the αγ and βγ complexes depends primarily on hydrophobic interactions of amino acids located in the second half of the coiled-coil region of the molecule. These interactions occur in the rough endoplasmic reticulum in the presence of various chaperones such as BiP.

A rapid enzyme-linked assay for ADAMTS-13

Summary.  Background: A deficiency in the plasma metalloprotease ADAMTS‐13 is associated with deposition of microvascular thrombi that cause thrombotic thrombocytopenic purpura. Current assays for ADAMTS‐13 are technically complex and time‐consuming. The objective of this study is to devise a rapid and sensitive assay for ADAMTS‐13 activity in plasma and verify the site of cleavage. Method: A new enzyme‐linked substrate, which contains a core ADAMTS‐13‐specific peptide conjugated to horseradish peroxidase (HRP) at the N‐terminus, and labeled with biotin at the C‐terminus, was constructed. After cleavage of this substrate by plasma ADAMTS‐13 and removal of uncleaved substrate by adsorption with streptavidin–agarose, ADAMTS‐13 activity was quantitated by determining the unadsorbed HRP activity remaining in solution. Levels of inhibitory antibodies in test plasma were also determined by measuring the residual ADAMTS‐13 activity after varying amounts of test plasma were incubated with a known amount of ADAMTS‐13. Results: Plasma ADAMTS‐13 activity was readily determined in ∼60 min (coefficient of variation 5.8%) using 1 μL of test plasma. Amino acid sequencing of the cleavage product confirmed that cleavage occurred at the Tyr1605‐Met1606 bond in the substrate. ADAMTS‐13 activities in the plasma of five TTP patients were below 2%. Inhibitory antibody titers in these samples varied from undetectable to 81 BU mL−1. Conclusion: The HRP‐linked substrate provides a rapid, sensitive, and reproducible way of determining the levels of ADAMTS‐13 activity and inhibitory antibodies in plasma.