Effect of phosphorylation in vitro of human fibrinogen with protein kinase C on thrombin-induced gelation

Thrombin-induced gel formation of fibrinogen phosphorylated by protein kinase C yielded a transparent gel, whereas unphosphorylated fibrinogen yielded a coarse gel. The mass-length ratio was found to be one order of magnitude higher for the unphosphorylated than for the phosphorylated fibrinogen. Since the phosphorylated sites are located near the cross-linking sites in the A alpha-chain of fibrinogen, it is likely that the introduction of charged phosphate groups in this region prevent the lateral growth of the fibrin fibres.     

Dephosphorylation of human fibrinogen, previously phosphorylated in vitro by protein kinase C, by whole blood or intestinal alkaline phosphatase. Effects on thrombin-induced gelation of in vitro dephosphorylated human fibrinogen

Human fibrinogen, a phosphoprotein, was either left untreated or phosphorylated by protein kinase C. Then both were dephosphorylated by calf intestinal alkaline phosphatase. The dephosphorylated fibrinogen gave an increased fibre thickness during thrombin-induced gelation. Whole blood anticoagulated by heparin, EDTA or sodium citrate, contained dephosphorylating activity against 32P-labeled fibrinogen, although there were significant differences in activity among the three anticoagulants.     

Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D

Prolonged degradation of fibrinogen by plasmin at calcium chloride concentrations of about 2 mM yields one major fragment D, designated as D(cate)¹. This fragment has a MW of 93,000 as derived from the MW of its subunits of 12,000, 38,000 and 43,000. Calcium ions protect D(cate) against further attack by plasmin. After sequestration of calcium ions with EGTA, fragment D(cate) is further degraded to a final fragment D, designated as D(EGTA), with a MW of 80,000 by breakdown of the γ-chain subunit⁷ 38,000 MW to 25,000 MW. The varying degrees of degradation of D(cate) in a medium containing no calcium or only traces may account for most of the $\text{in vitro}$ heterogeneity of fibrinogen fragment D described in the literature. Similarly, degradation of non-crosslinked fibrin by plasmin depends on the calcium concentration. Prolonged incubation of crosslinked fibrin with plasmin at 2 mM calcium chloride concentration resulted in the formation of a fragment D-dimer, with subunits of MW of 12,000, 43,000 and 75,000, indicating that it is comprised of two molecules of D(cate) which are crosslinked by the γ-subunit of 38,000 MW. The D-dimer also appeared to be plasmin-resistant by virtue of the presence of calcium ions. Both D(cate) and the D-dimer formed at calcium concentrations normally occurring in blood can be expected to represent physiologically important fragments D.     

The effect of fibrinogen degradation products and some lysine analogues on the dissociation of plasmin(ogen) - fibrin complexes

Fibrin carries sites which bind plasmin and preactivated plasminogen (mol.wt. 84,000) allowing the formation of stable complexes. Fibrinogen degradation product E interferes with this complex formation suggesting that the plasmin(ogen) binding site is present in this fragment. The complexes are also dissociated by the anti-fibrinolytic agents 6-aminohexanoic acid and aminomethyl cyclohexanoic acid which both compete for the fibrin binding site of plasmin and plasminogen.     

Effect of soluble and immobilized plasmin on fibrinogen and platelets

Plasmin was immobilized on collageno-elastic tubes (CET) using carbodiimide as the cross-linking agent. The effects of plasmin-CET grafts and corresponding soluble plasmin on fibrinogen, thrombin-mediated fibrinogen activation, and platelet activity, were investigated. There was a significant increase in fibrinogen deposition on plasmin-CETs over non-plasmin (i.e. control) CETs. Furthermore, exposure of fibrinogen to plasmin CETs enhances its deposition to control grafts situated downstream. Plasmin-bound CETs retained higher platelet deposition when preliminarily coated with fibrinogen. Finally, plasmin exerted a positive effect on thrombin-mediated fibrinogen activation at low plasmin concentrations. A mechanistic hypothesis aimed at interpreting this finding is proposed.     

The accelerating effect of fibrinogen and early fibrinogen degradation products on erythrocyte sedimentation

The fibrinogen molecule has a number of biological activities and some of these are shared by the larger degradation products. This study was carried out to investigate the erythrocyte sedimentation-accelerating property of fibrinogen and how this property might be modified by proteolytic digestion with plasmin. The sedimentation rate of washed human red cells suspended in saline, was found to be directly proportional to the concentration of added purified human fibrinogen, down to 250 mg%, below which there was no difference from saline controls. Plasmic digestion of fibrinogen yielding fragment X, did not reduce the accelerating affect on erythrocyte sedimentation, indicating that the intact carboxyl terminal end of the A alpha chain is unnecessary for this phenomenon. Further digestion to fragment Y reduced the effect slightly but digestion to fragments D and E abolished the accelerating effect completely.     

A simple and practical method for isolation of an early plasmin degradation product of human fibrinogen.

One of the earliest plasmin degradation products of human fibrinogen, so-called fragment A, was isolated by a simple method. This peptide has a molecular weight of approximately 22,500, migrating electrophoretically at beta-area, and its amino acid composition shows a very high content of glycine, serine, threonine and proline, and a markedly low content of hydrophobic amino acids. This fragment does not react against anti-fibrinogen; however, the anti-serum of this fragment reacts strongly with fibrinogen.     

Fibrinogen Haifa: fibrinogen variant with absence of protective effect of calcium on plasmin degradation of gamma chains

The abnormal fibrinogen Haifa is characterized by the fact that calcium present during enzymatic digestion by plasmin does not protect the Haifa D gamma chain against further plasmin attack as it does in normal molecules. Since calcium binding to fibrinogen, ADP--platelet aggregation cofactor activity and gamma dimerization process induced by factor XIIIa are normal for fibrinogen Haifa, the corresponding sequences in the gamma chain are not involved. It seems rather that the anomaly resides near the gamma 302 plasmin cleavage site that is protected when calcium is bound to the gamma chain and that this affects the availability of the polymerization site located in the C terminal part of the chain.     

Fibrinogen subunit degradation in vitro: studies on human fibrinogen heterogeneity.

A quantitative procedure for following fibrinogen subunit degradation $\text{in vitro}$ or $\text{in vivo}$, by measuring the loss of radioactivity from ¹²⁵I-fibrinogen, was used to determine the fibrinogenolytic capacity of human plasma. The results show that normal plasma in the absence of added activators does not have the ability to degrade fibrinogen $\text{in vitro}$. This observation was made using ¹²⁵I-labelled heterogenous fibrinogen and also with labelled low and high solubility forms of fibrinogen. The levels of plasminogen activator and plasmin required to cause fibrinogenolysis are higher than the reported levels for normal plasma. The relative labelling of the Aα, Bβ and γ subunits of low and high solubility fibrinogen were correlated with the reported loss of peptide from the subunits.     

Granulocyte enzyme mediated degradation of human fibrinogen in plasma in vitro

Incubation of plasma with granulocyte enzymes in the presence of kallikrein inhibitor resulted in a prolonged thrombin time of the plasma and in an increased level of fibrinogen fragments, indicating that fibrinogen was digested. In accordance with this, fibrinogen digestion products could be purified by affinity chromatography from plasma after incubation with granulocyte enzymes. The isolated products resembled early X-like fibrinogen fragments, which are produced by limited digestion of purified fibrinogen with elastase. On SDS gel electrophoresis both had no intact Aα-chains, but apparently intact fibrinogen Bβ- and γ-chains. Also, both fragments isolated from plasma and the X-like fragments produced with purified elastase had a low anticoagulant activity. Although elastase, the main fibrinolytic enzyme of the granulocyte, was rapidly complexed with inhibitors, 10–20% of the elastase activity towards succinyl-trialanyl-paranitro-anilide was detectable in plasma no matter if the mixture of granulocyte enzymes or purified elastase had been added. A possible role for the α₂-macroglobulin-granulocyte elastase complex in the production of the digestion products in plasma is discussed.     

日本刺沙蚕纤溶酶体内降纤作用与体外溶栓作用研究

目的研究日本刺沙蚕纤溶酶体内降纤及体外溶栓作用。方法通过一次性静脉给药后不同时间测定家兔纤维蛋白原含量,观察该酶的体内降纤作用;通过在体外测定对不同时间血栓的溶解百分率及D-二聚体含量,观察该酶体外血栓溶解作用;通过SDS-PAGE观察该酶对纤维蛋白原的水解作用机制。结果日本刺沙蚕纤溶酶可通过水解纤维蛋白原而降低体内纤维蛋白的含量;日本刺沙蚕纤溶酶在体外有显著的溶解血栓作用。结论日本刺沙蚕纤溶酶具有体内降纤及体外溶栓作用。     

Analysis of reptilase and thrombin-induced changes in fibrinogen subunits by isoelectric focussing

Isoelectric focussing (IEF) in 6M urea/4% polyacrylamide gel of reduced human fibrinogen and the fibrins formed by Reptilase and thrombin gave a complex multibanded pattern. Similar results were obtained on IEF in urea/agarose gels containing excess mercapto-ethanol. To characterize these multiple bands on the basis of molecular weight secondary electrophoreses into a discontinuous SDS-polyacrylamide gel system were carried out perpendicular to the initial IEF's. At least five major Alpha chains ranging in pI from 7.14 to 7.60 were found in fibrinogen. These were shifted 0.40 pI units towards the cathode after the action of Reptilase or thrombin due to the release of electronegative fibrinopeptide A species. A similar change occurred with the three chain B beta chains with initial pI's of 6.81 to 7.13, but only after thrombin action. Gamma dimer showed no significant difference in pI from its component chains 6.05 to 6.17.     

Digestion of amidinated and phenylglyoxal treated fibrinogen by plasmin.

Human fibrinogen with the lysyl and/or arginyl residues modified by treatment with ethyl acetimidate hydrochloride and phenylglyoxal monohydrate, respectively, was incubated with plasmin. Both chemical modifications delayed the appearance of components Y, D and E while component X was not affected.     

The generation of chemotactic activity for human leukocytes by the action of plasmin on human fibrinogen.

Chemotactic activity for human peripheral blood leukocytes was generated by the action of plasmin on human fibrinogen. When plasmin digestion was stopped at time intervals up to 24 hours, a small amount of activity was present at 15 and 30 min. corresponding to the transient appearance of fragment Y. The activity contained in the 24-hour digest was considerably higher and eluted from Sephadex G-75 with molecules of approximately 30,000 daltons. When purified X, Y, D and E were assayed individually for chemotaxis only fragment Y was active but in relatively high concentrations. Thus the chemotactic activity generated by the action of plasmin on fibrinogen was mainly associated with one or more lower molecular weight polypeptides and to a lesser extent with the Y fragment.     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

The effects of dephosphorylation on the structure of the projections of neurofilament

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.     

Studies of the advanced stages of the plasmin and trypsin digestion of bovine fibrinogen

The advanced stages of the digestion of bovine fibrinogen by trypsin and by plasmin have been investigated. The fragmentation of the molecule has a nearly identical course with the two enzymes, however, plasmin accomplishes the same result by cleaving only 1/2 to 2/3 as many bonds as trypsin. The advanced digestion can be divided into two stages. First, fragment D formed during the early part of the reaction is transformed into a number of successively lower molecular weight subspecies, with accompanying liberation of peptides of lower than 16,000 molecular weight. Second, in a very slow process, the products of the preceding stage are broken up into a homogeneous mixture of fragments encompassing a wide range of molecular weights. Fragment E remains unchanged during the whole process.