BINDING OF NATIVE PLASMINOGEN TO FIBRIN AND TO SOME FI BRINOGEN/FIBRIN DERIVATIVES

1987 ◽  
Author(s):  
C Zamarrón
Keyword(s):  
Cyanogen Bromide ◽  
Dissociation Constants ◽  
Solid Phase ◽  
Caproic Acid ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Equilibrium Conditions ◽  
Fibrin Monomer ◽  
Rate Conversion ◽  
Determinant Factor

In the fibrinolytic process: (a) fibrin provides a surface on which the major reactions of fibrinolysis occurs: the conversion of plasminogen to plasmin, the cleavage of fibrin by plasmin and the inhibition of plasmin by α2-antiplasmin, (b) some fibrinogen derivatives (e.g. the cyanogen bromide digested fibrinogen fragment denominated FCB-2) can exert stimulatory properties on the plasminogen activation and (c) the initial cleavage of fibrin by plasmin increases the rate conversion of plasminogen to plasmin.The purpose of the present work has been to correlate these three aspects of the fibrinolytic process with the binding of native plasminogen (Glu-Pg) to fibrin (Fn) , fibrinogen (Fg) and Fn/Fg derivatives.The Glu-Pg-Fg interaction, if exists, it is not detectable in equilibrium conditions by analytical centrifu gation. By using a solid-phase fibrin clot system (purified system) the Glu-Pg-Fn interaction gives the following dissociation constants: Kd=3.5×10−6 M and 1.2×10−5 m (unwashed and washed clots respectively). Being two the number of plasminogen binding sites per fibrin fibrin monomer. By activation with streptokinase or urokinase the amount of Pg required for an effective lysis of the fibrin clots is lower when the Pg is endogenous (inside the clot) versus exogenous (outside the clot).The binding of the isolated fragments of the cyanogen bromide digested fibrinogen to Glu-Pg was studied by affinity chromatography on Glu-Pg-Sepharose. The only fragment bound to Glu-Pg and eluted with 10 mM ε-amino caproic acid (ε;-ACA) was the fragment denominated FCB-2 The soluble fibrin monomer after 20 min plasmin digestion also binds to immobilized Glu-Pg and it is eluted with ε-ACA.Therefore, the binding of native plasminogen to fibrin and to some fibrinogen/fibrin derivatives is a determinant factor in the three aspects of the fibrinolytic process mencioned above.

scholarly journals The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 555-565
Author(s):  
V Gurewich ◽  
E Hyde ◽  
B Lipinski
Keyword(s):  
Plasminogen Activator ◽  
Solid Phase ◽  
Fibrin Clot ◽  
Reference Curve ◽  
Plasminogen Activation ◽  
Large Excess ◽  
Soluble Fibrin ◽  
Alternative Pathways ◽  
Fibrin Monomer ◽  
Fibrin Plate

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.

scholarly journals The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 555-565 ◽  
Author(s):  
V Gurewich ◽  
E Hyde ◽  
B Lipinski
Keyword(s):  
Plasminogen Activator ◽  
Solid Phase ◽  
Fibrin Clot ◽  
Reference Curve ◽  
Plasminogen Activation ◽  
Large Excess ◽  
Soluble Fibrin ◽  
Alternative Pathways ◽  
Fibrin Monomer ◽  
Fibrin Plate

Abstract The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.

Fibrin Formation: The Role of the Fibrinogen-Fibrin Monomer Complex

1976 ◽  
Vol 36 (01) ◽  
pp. 037-048 ◽  
Author(s):  
Eric P. Brass ◽  
Walter B. Forman ◽  
Robert V. Edwards ◽  
Olgierd Lindan
Keyword(s):  
Particle Size ◽  
Induction Period ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Appreciable Amount ◽  
Buffer System ◽  
Homeostatic Mechanism ◽  
Fibrin Formation ◽  
Fibrin Monomer

SummaryThe process of fibrin formation using highly purified fibrinogen and thrombin was studied using laser fluctuation spectroscopy, a method that rapidly determines particle size in a solution. Two periods in fibrin clot formation were noted: an induction period during which no fibrin polymerization occurred and a period of rapid increase in particle size. Direct measurement of fibrin monomer polymerization and fibrinopeptide release showed no evidence of an induction period. These observations were best explained by a kinetic model for fibrin clot formation incorporating a reversible fibrinogen-fibrin monomer complex. In this model, the complex serves as a buffer system during the earliest phase of fibrin formation. This prevents the accumulation of free polymerizable fibrin monomer until an appreciable amount of fibrinogen has reacted with thrombin, at which point the fibrin monomer level rises rapidly and polymerization proceeds. Clinically, the complex may be a homeostatic mechanism preventing pathological clotting during periods of elevated fibrinogen.

scholarly journals RESEARCH OF AROMA FORMED COMPONENTS OF RENNET CHEESES

Food systems ◽  
2019 ◽  
Vol 1 (4) ◽  
pp. 19-26
Author(s):  
I M. Pochitskaya ◽  
A. P. Laktionova ◽  
V. L. Roslik
Keyword(s):  
Gas Chromatography ◽  
Acetic Acid ◽  
Solid Phase ◽  
Fiber Material ◽  
Caproic Acid ◽  
Volatile Components ◽  
Selective Detection ◽  
Hexyl Acetate ◽  
Main Components ◽  
Flavor Profile

The results of the study of the composition of volatile compounds of rennet cheeses are presented. Sample preparation was performed using Supelco’s solid-phase microextractor, including a special fiber material coated with a layer of divinylbenzene-carboxene-polydimethylsiloxane «DVB / Carboxen / PDMS StableFlesh ™». Using gas chromatography with mass-selective detection, about 400 aroma-forming components were detected, among which 39 compounds were identified, which form the basis of the flavor profile of cheeses. The main flavor descriptors for all cheeses are: propionic, butyric, and caproic acids, acetoin, methyl amyl ketone, 2-nonanone, and limonene. It has been established that the profile of volatile components for cheeses various ripening periods, has significant differences. Thus, for semi-hard cheeses made using propionic acid microorganisms, the most characteristic components are acids — propionic and butyric, as well as compounds of the terpene series o-cymene and β-pinene. For semi-hard cheeses of the Dutch group (formed from the layer), with maturities from 20 days to 3 months, the terpene compounds and esters are the most significant, whereas for superhard cheeses with a maturity of more than a year, the main components affecting the aromatic profile are butyric and caproic acids, 2-heptanone and limonene. Semi-hard and hard cheeses with ripening periods from 3 to 6 months are characterized by a high content of caproic and butyric acids, as well as by the presence of such aromatic substances as limonene and acetic acid ethyl acetate (hexyl acetate). Semi-hard cheeses, which are molded in bulk, with maturities from 10 days to 3 months contain acetoin, caproic acid, as well as significant quantities of the same limonene and hexylacetate.

A Direct, Plasmin Independent Assay For Plasminogen Activation

1981 ◽  
Author(s):  
Luciana Mussoni ◽  
Dan Lawrence ◽  
David Loskutoff
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Caproic Acid ◽  
Plasminogen Activation ◽  
Single Chain ◽  
New Approach ◽  
Aortic Endothelial Cells ◽  
Cleavage Assay ◽  
Km Value ◽  
Triton X ◽  
Dose Dependent

We have modified the direct, 125I-plasminogen cleavage assay for plasminogen activator (PA) and employed it to compare urokinase (UK), tissue activator (TA), and PAs produced by cultured bovine aortic endothelial cells. The assay is based on conversion of single chain plasminogen to two chain plasmin as revealed by polyacrylamide gel electrophoresis in the presence of SDS andβ-mercapto- ethanol. Inclusion of Triton X-100, albumin and trasylol in the reaction mixture reduced the adsorptive and hydrolytic loss of reactants, and increased the linearity and sensitivity of the assay. Under these conditions, plasmin formation was linear for at least 6 hrs, dose- dependent over a 20-fold range of UK concentrations, and at least 100-fold more sensitive (0.01 units/ml) than previously reported direct assays for UK. In preliminary experiments, we determined a Km value of 10yM for UK and plasminogen. Activation of plasminogen by TA was minimal in the absence of fibrin, and independent of the concentration of activator. However, in the presence of fibrin, (1) the initial rate of activation increased dramatically, (2) 100-1000 fold less TA was required, and (3) activation was proportional to the concentration of both TA and fibrin. Surprisingly, activation by UK and cellular PAs was partially inhibited by fibrin. Epsilon amino caproic acid (EACA; 0.5-100mM) stimulated activation by UK both in the presence and absence of fibrin by 30-40%. In contrast, EACA (0.1-100mM) inhibited TA activity in the presence of fibrin by over 90%. However, in the absence of fibrin, TA was inhibited by only 50%, even at high EACA concentrations (lOOmM). These results indicate that cleavage of 1251- plasminogen can be employed as a direct, sensitive and quantitative assay for various PAs, and offers a new approach for studying plasminogen activation and agents that stimulate or inhibit it.

L-Lysine: A Modulator for the Relative Activity of High Molecular Weight (HMW) and Low Molecular Weight(LHW) Urokinase(UK)

1979 ◽  
Author(s):  
L.L. Shen ◽  
W.H. Holleman
Keyword(s):  
Molecular Weight ◽  
Caproic Acid ◽  
Fibrin Clot ◽  
Relative Activity ◽  
Test Tube ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Plasma Clot ◽  
Concentration Dependent Manner ◽  
The Difference

L-Lysine(Lys), in a concentration dependent manner, progressively inhibited UK-activated lysis of human plasma clots as demonstrated by Ploug test-tube method and elastometric measurements. Lys was more effective with HMW UK than LMW UK, and the effect of Lys with LMW UK from tissue culture and urine sources was the same. Epsilon amino caproic acid(EACA) and tranexamic acid(TXA) were stronger inhibitors but inhibited HMW and LMW UK-induced lysis to the same degree. Elastometric measurements showed that Lys inhibition was not due to its interference with the initial clotting process nor to the reduction of clot rigidity. Amidolytic assays using chromogenic substrates showed that Lys had no direct effect, on UK, and that Lys enhanced the activation of the native Glu-plasminogen(Pg) by LMW UK, but not the activation by HMW UK. When the substrate was human fibrin clots, Lys enhanced the lysis induced by LMW UK while the lysis induced by HMW UK was inhibited; however, the extent of enhancement and inhibition was limited. We concluded that the mode of Lys action is not identical to that of EACA or TXA, and that the stronger Lys inhibition of plasma clot lysis as compared to fibrin clot lysis is due to the potentiation of plasma fibrinolytic inhibitors by Lys. The difference In effect of Lys on HMW and LMW UK-induced lyels is likely due to a partial conformation change of Glu-Pg molecule upon Lys binding. The relatively moderate interaction of Lys with Glu-Fg results In a mildly modified UK substrate which reacts preferentially with the enzyme smaller in size.

FIBRINOGENS NEW YORK II, III, AND IV : RELATIONSHIP BETWEEN ABNORMAL FIBRIN MONOMER POLYMERIZATION, BLEEDING TENDENCY, AND THROMBOTIC TENDENCY

1987 ◽  
Author(s):  
George Karp ◽  
Chung Y Liu
Keyword(s):  
New York ◽  
Normal Level ◽  
Molecular Size ◽  
Plasma Fibrinogen ◽  
Bleeding Tendency ◽  
Plasminogen Activation ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Peptide Release ◽  
Thrombotic Tendency

It has been proposed that a normal fibrin monomer polymerization is required for a normal fibrin potentiation of tPA-induced plasminogen activation. Accordingly, an abnormal fibrin moncmer polymerization may contribute to a thrombotic tendency. To investigate this possibility, three abnormal plasma fibrinogens (dysfibrinogenemia) have been studied and tentatively designated New York II (NY-II), New York III (NY-III), and New York IV (NY-IV) after the city where they were described.Each of these patients has a long thrombin clotting time (TCT), a long Reptilase clotxing time, and an abnormal fibrin monomer polymerization. The abnormal fibrin moncmer polymerization can be corrected by addition of Ca2+. NY-II is characterized by a normal level of plasma fibrinogen, but delayed fibrino-peptide release and marked prolongation of the TCI; NY-III, by a normal level of plasma fibrinogen and a normal fibririopeptide release; and NY-IV, by a low level of plasma fibrinogen, a normal fibrinopeptide release, and marked prolongation of the TCT. Each of these cases appears to be congenital, not acquired although the absence of data on relatives prevents definite conclusions. SDS-PAGE analysis of the mobility of the reduced abnormal fibrinogens shows no detectable abnormality, indicating no significantly altered molecular size of Aα, Bβ, and γ chains.No patient appears to be associated with either a bleeding or a thrombotic tendency. Thus, no direct association is observed between an abnormal fibrin monomer polymerization and a thrombotic tendency. Accordingly, fibrin monomer polymerization is not a function absolutly required for potentiation of plasminogen activation. However, based on the present observations and the observations previously described of abnormal fibrinogens with abnormal fibrin monomer polymerization from patients associated with bleeding and/or thrombotic tendency, a bleeding tendency seems to be correlated with a condition in which both a long TCT and an abnormal fibrin moncmer polymerization can not be normalized by addition of Ca2+.

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4380-4386 ◽  
Author(s):  
Krasimir Kolev ◽  
Kiril Tenekedjiev ◽  
Katalin Ajtai ◽  
Ilona Kovalszky ◽  
Judit Gombás ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Rate Constant ◽  
Dissociation Constants ◽  
Dissociation Rate ◽  
Covalent Modification ◽  
Tissue Type ◽  
Factor Xiiia ◽  
Molar Ratio ◽  
Fibrin Monomer

Abstract Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 μM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 × 102 M–1 · s–1 and a dissociation rate constant of 3.07 × 10–4 s–1 are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosinfibrin clot is relatively resistant to plasmin.

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