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Biomedical subjects

C Kluft

Publications and source records attributed to C Kluft.

At least 199 records · Page 11Linked to original sources
PubMed1989

Histidine-rich glycoprotein is elevated in mild liver cirrhosis and decreased in moderate and severe liver cirrhosis.

Histidine-rich glycoprotein (HRG) reduces by complex formation with plasminogen the amount of "free" plasminogen in circulation and is therefore considered an inhibitor of fibrinolysis. We studied the levels of both HRG and plasminogen in patients with different degrees of liver cirrhosis to assess the role of HRG in enhanced fibrinolysis in these patients. In mild (Child A) cirrhosis, HRG levels unexpectedly were significantly increased. The total plasminogen level and the calculated amount of free plasminogen were decreased. In moderate (Child B) cirrhosis, both HRG levels and total plasminogen levels were reduced, resulting in a normal amount of free plasminogen in circulation. In severe (Child C) cirrhosis, HRG level, total plasminogen level, and free plasminogen level were all decreased. Because the HRG level is increased in Child A liver cirrhosis, we suggest that other mechanisms, other than simply a decreased synthetic capacity of the liver, contribute to the changes in HRG levels in patients with liver disease. Because of the reduction of free plasminogen levels in severe liver cirrhosis, we propose that the decrease in HRG levels in liver cirrhosis plays no role in enhanced fibrinolysis in these patients. The increase in the HRG level in Child A liver cirrhosis may be of importance in future studies on familial thrombosis associated with elevated levels of HRG, where mild liver dysfunction should at least be excluded.

Adult↗
PubMed1988

Modified crossed immunoelectrophoresis to study with whole plasma the reversible complex formation of histidine-rich glycoprotein with plasminogen.

To study the reversible complex formation between the plasma protein histidine-rich glycoprotein (HRG) and plasminogen, crossed immunoelectrophoresis of HRG was modified. In the modification, purified plasminogen was introduced into the gel of the first dimension electrophoresis. Two molecular forms of plasminogen, Glu- and Lys-plasminogen, induced a dose-dependent reduction of the electrophoretic mobility of HRG, with a half maximal retardation for both plasminogens at 0.50-0.55 microM of added plasminogen to the agarose gel. HRG in plasma behaved as a uniform fraction with respect to plasminogen binding. In contrast, with the same modified technique another plasma protein, alpha 2-antiplasmin, separated into a retarded plasminogen-binding form and a non-retarded non-plasminogen-binding form. The method can be used to assess several aspects of reversible complex formation between plasma proteins, as demonstrated for plasminogen binding of HRG and alpha 2-antiplasmin in whole plasma.

Binding, Competitive↗
PubMed1988

Acid treatment of plasma for the inactivation of plasminogen activator inhibitor-1 (PAI-1).

The present study was initiated to assess the effectiveness of various acid treatments of blood or plasma in the inactivation of PAI-1. It was shown that a frequently used treatment of blood or plasma with 1 M acetate buffer, pH 3.9, only partially inactivated PAI-1. The inactivation of PAI-1 in plasma was found to depend upon pH and temperature, showing an optimal inactivation at a pH less than or equal to 3, at 37 degrees C.

Acetates↗
PubMed1988

Alpha 2-antiplasmin Enschede is not an inhibitor, but a substrate, of plasmin.

alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.

Amino Acid Sequence↗
PubMed1988

Daytime fluctuations in blood of tissue-type plasminogen activator (t-PA) and its fast-acting inhibitor (PAI-1).

Circadian fluctuation in blood fibrinolytic activity was studied in 10 volunteers in the day-time period at 09.00, 12.00 and 15.00 h. Activity of tissue-type plasminogen activator (t-PA) was found to increase from 09.00 to 15.00 h in accordance with known results with global assays of blood activity. Also, activity and antigen of plasminogen activator inhibitor 1 (PAI-1) and antigen of t-PA showed fluctuations but with an opposite pattern to that of t-PA activity, with highest levels in the morning and lowest ones in the afternoon. Activity of a reversible t-PA inhibitor was constant. The discordance between fluctuations in antigen and in activity of t-PA is attributed to the inhibitory effect of PAI-1 on t-PA activity. It is concluded that the appearance into the blood of t-PA and PAI-1 shows a daytime fluctuation with a similar pattern, suggesting a co-ordinated circadian fluctuation in production of both proteins by endothelial cells.

Adult↗
PubMed1988

Mild haemostatic problems associated with congenital heterozygous alpha 2-antiplasmin deficiency.

A Dutch family, of which 13 members are heterozygotes, deficient for alpha 2-antiplasmin (alpha 2-AP) is reported. Clinical studies showed that 2 heterozygotes had a mild bleeding tendency, which presented as bleeding episodes after tooth extraction and after surgery and, in one patient, also as excessive menstruation. Laboratory investigations revealed an alpha 2-AP activity of 62% (51-71) (median and range) and an antigen level of 60% (60-66). The plasminogen binding as well as the fibrin binding properties of alpha 2-AP were normal. Plasminogen concentrations were significantly higher in the heterozygotes compared to the other family members. However, free plasminogen not bound to histidine-rich glycoprotein was not significantly different between these two groups. We propose that in this family the deficiency of alpha 2-AP is due to a decreased synthesis of a normal alpha 2-AP molecule. This present study brings the frequency of heterozygous alpha 2-AP deficient patients with a bleeding tendency to 13 out of 59 heterozygotes reported in the literature.

Adult↗
PubMed1988

Haemostasis contact system and fibrinolysis in hereditary angioedema (C1-inhibitor deficiency).

Factors of the classical complement pathway, the contact system and fibrinolysis were evaluated both with functional and immunochemical methods, in patients with inherited deficiency of C1-inhibitor. Evaluations were performed under basal conditions, during acute attacks and during prophylaxis with low doses of anabolic steroids. Patients in the basal state showed no significant abnormalities of any of the parameters that we investigated. During acute attacks a slightly reduced prekallikrein concentration was registered. During treatment with low doses of danazol and stanozolol, protein C and plasminogen were found to be increased. Our data suggest that C1-inhibitor deficiency per se does not lead to a derangement of the fibrinolysis and coagulation contact system, and that the kinin system may be involved during acute attacks of angioedema.

Adolescent↗
PubMed1988

[Recombinant tissue-type plasminogen activator: pharmacokinetics and effect on the hemostasis system of the human].

The in vitro interaction of rt-PA with the coagulation and fibrinolytic system was investigated. After addition of rt-PA to citrated plasma prolonged clotting times for prothrombin time, partial thromboplastin time and thrombin time, decreased activities of individual clotting factors and decreases of alpha 2-antiplasmin, plasminogen and fibrinogen were measured. The alterations were dependent on incubation times and rt-PA concentrations and were mainly observed at rt-PA concentrations exceeding therapeutic levels. These in vitro phenomena could be prevented to different degrees using various inhibitors. Because of interferences with some fibrinolytic and coagulation assays aprotinin was only of limited utility as an inhibitor. PPACK lengthened clotting time assays based on generation of endogenous thrombin, but did not affect fibrinolytic assays. The most versatile inhibitor was a specific anti-rt-PA antibody which enabled correct measurements of all fibrinolytic and most coagulation assays. It is concluded that high rt-PA levels in samples taken from patients during fibrinolytic therapy induce in vitro artefacts which can be prevented by the use of suitable inhibitors. In 8 healthy male volunteers aged 32 +/- 7 years pharmacokinetic investigations were done. After infusion of 0.25 mg rt-PA/kg bw mean maximal plasma levels were 970 +/- 130 ng/ml. The elimination of rt-PA from plasma was fitted to a two compartment model and was characterized by two half lives of t1/2 alpha = 3.3 and t1/2 beta = 26 minutes.

Adult↗
PubMed1988

Fibrinolytic agents and their effects on the haemostatic system.

Fibrinolytic agents used in intravenous thrombolytic therapy of myocardial infarction also exert, at effective dosages, significant side effects on the haemostatic system outside the direct resolution of the target thrombus. The side effects of the agents streptokinase and anisyolated plasminogen-streptokinase activator complex are larger than the side effects of the more fibrin-specific agents tissue-type plasminogen activator and pro-urokinase. An important difference between the two groups of compounds is that the major plasmin inhibitor alpha 2-antiplasmin is completely exhausted for the first two agents, but only in part of the cases for the fibrin-specific agents. The effects on various factors in the fibrinolytic cascade on the plasminogen activator, plasminogen-plasmin and fibrinogen-fibrin levels are reviewed concisely.

Blood Coagulation Tests↗
PubMed1988

Pharmacokinetics of antigen and activity of recombinant tissue-type plasminogen activator after infusion in healthy volunteers.

Pharmacokinetics of recombinant tissue plasminogen activator (rt-PA, large-scale process) were determined based on antigen and activity after infusion of 0.25 mg/kg in 8 healthy volunteers. Plasma antigen was measured using enzyme-linked immunosorbent assay (ELISA) with and without treatment of blood at collection with D-Phe-Pro-Arg-CH2Cl (P PACK); activity was quantified in acidified plasma both on fibrin plates and in a chromogenic assay. Highest rt-PA concentrations were measured in ELISA with P PACK-treated samples, yielding the following pharmacokinetic parameters (2-compartment model, mean +/- S.D.): Cmax = 973 +/- 133 ng/ml, CL = 687 ml/min, dominant half-life t1/2 alpha = 3.3 +/- 0.4 min, t1/2 beta = 26 +/- 12 min, V1 = 3.9 +/- 0.6 l and Vss = 7.2 +/- 1.0 l. The other assays yielded lower rt-PA concentrations, which affected clearance and volume parameters but not t1/2 beta and t1/2 beta. Linear regressions of the fibrin plate and chromogenic assay results vs. ELISA yielded excellent correlations (R greater than 0.96, n = 55-57) but slopes of 0.76 and 0.64, respectively. This indicates that about 25-35% of rt-PA antigen in thawed plasma samples are not detected in activity assays, due at least partially to in vitro binding of rt-PA by proteinase inhibitors.

Chromogenic Compounds↗
PubMed1987

Depression of tissue plasminogen activator (t-PA) activity and rise of t-PA inhibition and acute phase reactants in blood of patients with acute myocardial infarction (AMI).

We determined during the acute stage of myocardial infarction selected fibrinolysis variables (tissue-type plasminogen activator, intrinsic plasminogen activators, tissue-type plasminogen activator inhibition, C1-inactivator) and related the observed changes to changes in two acute phase reactants (C-reactive protein, fibrinogen). Acute myocardial injury induce significant increases in blood of tissue-type plasminogen activator inhibition (day one, p less than 0.05), C-reactive protein (day three, p less than 0.01), fibrinogen (day six, p less than 0.01), and C1-inactivator (day eight, p less than 0.01). Tissue-type plasminogen activator activity measured as C1-inactivator resistant fibrinolytic activity showed a minimum day two after the acute attack (p less than 0.01), whereas plasminogen activator activities arising from the intrinsic system of fibrinolysis remained constant. The observed changes did not parallel the occurrence of deep vein thrombosis indicated by a positive Tc-plasmin test (41% of the patients).

Acute-Phase Proteins↗
PubMed1987

Alpha 2-antiplasmin Enschede: alanine insertion and abolition of plasmin inhibitory activity.

An abnormal alpha 2-antiplasmin that is associated with a serious bleeding tendency has been found in a Dutch family and is referred to as alpha 2-antiplasmin Enschede. This abnormal alpha 2-antiplasmin is converted from an inhibitor of plasmin to a substrate. The molecular defect of alpha 2-antiplasmin Enschede, as revealed by sequencing of cloned genomic DNA fragments, consists of an alanine insertion near the active site region of the molecule. Substitution of this fragment into complementary DNA for a wild-type alpha 2-antiplasmin yields a translation product with physical and functional properties typical of the abnormal alpha 2-antiplasmin Enschede. The naturally occurring mutant may serve as a model for investigating the structures that determine the properties of an inhibitor versus those of a substrate in serine protease inhibitors.

Amino Acid Sequence↗