SummaryWe have produced a high-affinity monoclonal antibody classified as IgG1 with K-type light chains that recognizes the calcium ion(Ca2+)- dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca2+, the half-maximal binding being reached at 130 μM Ca2+. The dissociation constant was estimated to be 1.6 × 10-8 M at 2 mM Ca2+. The antibody was found also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the Aa chains depleted of the carboxy-terminal Aα(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.
Using urea-solubilized human fibrin monomer as an immunogen, we raised in mice a battery of monoclonal antibodies that reacted with the immunogen but not with urea-treated or native fibrinogen. Although they all failed to react with acid-solubilized fibrin monomer (acid-FM) alone, an antibody designated as IF-43 was found to recognize acid-FM, which was bound with fibrinogen or its derivatives to form a 1:2 complex of soluble fibrin. The epitope for this antibody, thus, appears to be exposed most probably by conformation changes induced in the acid- FM molecule upon formation of the complex. Because IF-43 was able to recognize fibrin-derived plasmic fragment E treated with urea but not the thrombin- and urea-treated amino-terminal disulfide knot of fibrinogen, the presence of the A alpha (52–78) residue segment seems to be prerequiste for the epitope expression. The antibody was found to react with soluble fibrin monomer spiked to normal plasma dose- dependently up to 200 micrograms/mL. By an aggregation assay using latex beads coated with IF-43, we found that concentrations of soluble fibrin monomer in plasma derived from patients with thrombotic diseases were mostly elevated, but not necessarily correlated with those of the D-dimer, reflecting another aspects of the disease. Furthermore, the soluble fibrin monomer in plasma derived from patients with thrombotic diseases was found to be depleted solely of the A peptides, but not the B peptides, based on its subunit polypeptide compositions lacking the beta-chain on immunoblotting.
To examine whether or not acquired alpha 2-plasmin inhibitor deficiency is associated with systemic fibrinogenolysis, we analyzed the fibrin and fibrinogen degradation products in eight patients with this condition in various disease states. The underlying disease was gastric cancer in three patients, metastatic prostatic cancer in two, acute promyelocytic leukemia in two, and abdominal aortic aneurysm in one patient. In all eight patients, the alpha 2-plasmin inhibitor level was reduced to less than 50% of normal, and plasmin-alpha 2-plasmin inhibitor complex levels were increased. Immunoblotting of serum using an antifibrinogen antibody detected a 250 kDa protein (corresponding to fragments X or DY) in all eight patients. Fragment Y and D monomer were detected in seven of the eight patients, indicating the occurrence of systemic fibrinogenolysis. However, they were not detected in one patient with metastatic prostatic cancer. To determine whether or not fibrinogen degradation was also occurring in the patient without fragment Y, we characterized the 250 kDa protein in all eight patients. The protein was found to be fragment X in the metastatic prostatic cancer patient without fragment Y, while it was fragment DY in the other seven patients. Thus, systemic fibrinogenolysis was present in all eight patients. In the two patients with metastatic prostatic cancer, the level of alpha 2-plasmin inhibitor gradually increased with the reduction of tumor size by treatment. Fragment X, fragment Y, and D monomer were not detected when the alpha 2-plasmin inhibitor level exceeded 60% of normal in both patients. In the other six patients fragment Y and D monomer also disappeared when the alpha 2-plasmin inhibitor level exceeded 60% of normal. These findings suggest that systemic fibrinogenolysis only occurs when the plasma levels of alpha 2-plasmin inhibitor falls below 60% of normal due to activation of the fibrinolytic system by various pathological conditions.
Human and murine fibroblasts were found to spread far more avidly on fibrin monomer monolayers than on immobilized fibrinogen, indicating that removal of fibrinopeptides by thrombin is a prerequisite for the fibrin-mediated augmentation of cell spreading. In fact, cell spreading was not efficiently augmented on monolayers of a thrombin-treated dysfibrinogen lacking the release of fibrinopeptide A due to an A␣ Arg-16 3 Cys substitution. Since a synthetic Arg-Gly-Asp (RGD)-containing peptide inhibited the fibrin-mediated cell spreading, subsequent dissociation of the carboxyl-terminal globular domain of the A␣-chains appears to render the RGD segments accessible to the cell-surface integrins. In support of this, fibrin-augmented cell spreading was inhibited by an antibody recognizing a 12-kDa peptide segment with ␥ Met-89 at its amino terminus, which is located in close association with the RGD segment at A␣ 95-97 in the helical coiled-coil interdomainal connector. The fibrin-mediated augmentation of cell spreading was inhibited not only by an antibody against human vitronectin receptor (LM 609) but also by an antibody against the  1 subunit of integrin (mAb13), suggesting that the  1 -class integrin together with a vitronectin receptor, ␣ v  3 , is mobilized onto the surface of fibroblasts upon contact with the fibrin monomer monolayer.Fibrinogen is a 340-kDa glycoprotein consisting of three pairs of polypeptide subunits, A␣, B, and ␥, linked together by multiple disulfide bonds (1). By structural studies including electron microscopic analysis together with biochemical data, there is now general agreement on the shape of the fibrinogen molecule (2-7). The fibrinogen molecule is composed of three major globular domains, i.e. one central E domain and two identical outer D domains connected by a three chain ␣-helical coiled-coil (4, 6). The distal part of the D domain is the carboxyl terminus of the ␥-chain, while the proximal part is the carboxyl terminus of the B-chain. The carboxyl-terminal two thirds of the A␣-chains fold back from the D domain and form two independently folded domains (␣C domains) at their carboxylterminal parts. In the native fibrinogen molecule, the ␣C domains interact with each other and form an additional small globular (␣C-␣C) domain that is closely associated with the central E domain (4, 6, 7). Upon thrombin cleavage of fibrinopeptides A and B, the globular ␣C-␣C domain is released from the E domain, and subsequently dissociated into individual ␣C domains (4, 6, 7). The fibrinogen molecule thus undergoes distinct conformational changes upon conversion to the fibrin monomer molecule (4), and thereby exposes several fibrin-specific regions that may participate in the functions of fibrin. The Arg-Gly-Asp (RGD) segments residing at A␣ 95-97 and A␣ 572-574, tentatively designated as RGD-1 and RGD-2, respectively, may also be categorized into this type of fibrin-specific segments. In this paper, we describe the binding of cultured human and murine fibroblasts to immobilized fibrin monome...
When granulocytes are stimulated under certain clinical conditions, elastase is released therefrom and digests fibrin(ogen) independently of the plasmin system, which may also be mobilized simultaneously. Thus, discrimination of these 2 systems becomes urgent for the diagnosis and treatment of the underlying diseases. Using as immunogen a 97-kd granulocyte-elastase digest of human fibrinogen, we raised an antibody IF-123 that specifically recognizes elastase digests of human fibrin(ogen). The 97-kd elastase fragment resembles plasmic fragment D1, and the epitope of this antibody is located on the A (196-204) residue segment. This segment appears to be masked in fibrin(ogen) but exposed when the A Leu 204-Ile 205 peptide bond is cleaved by elastase. Cathepsin G concomitantly released from granulocytes failed to expose the epitope. By an enzyme immunoassay using IF-123 as the capture antibody, the elastase digests of fibrin(ogen) can be measured in plasma samples without interference by abundantly coexisting fibrinogen. Indeed, we found that the elastase digests were mostly elevated in patients with inflammation or malignant tumors, but remained in a normal range in patients with a benign gastrointestinal tract disease such as duodenal ulcer and polyps in the gallbladder or the colon. Like the plasmic D-dimer, the elastase digests predominantly consisted of the DD/E complex and DD/E-containing high-molecular weight derivatives apparently corresponding to the phase-3 plasmic digests of cross-linked fibrin.
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