Biomedical subjects
Publications and source records attributed to C Kluft.
Various preparations of anti-urokinase antibodies were found to quench a part of the plasma fibrinolytic activity. This part was also inhibited by a specific, urokinase inhibitor isolated from placenta. These observations indicate that plasma contains a urokinase-related immuno-reactivity. This activity was found to belong to the factor XII-independent proactivator system.
Activation of prorenin in the neutral phase after pH 3.3 dialysis of human plasma depends on clotting factor XII-initiated prekallikrein to kallikrein conversion. Acid dialysis may be necessary for destroying kallikrein inhibitors or rendering prorenin susceptible to attack by kallikrein. If the latter possibility proves true, it is difficult to see how the factor XII-kallikrein pathway could activate prorenin in vivo. Plasma prorenin was therefore separated from active renin and from the protease inhibitors alpha 2-macroglobulin, C1-inactivator, alpha 1-antitrypsin, inter-alpha-trypsin inhibitor, and antithrombin III by gel filtration on Sephadex G-100 and affinity chromatography on Blue Sepharose CL-6B at neutral pH. The resulting prorenin preparation could be activated at pH 7.5 by highly purified human plasma kallikrein, which was prepared from prekallikrein by activation with active factor XII fragment beta-factor XII a. Activation proceeded at 4 and 37 C at a kallikrein concentration of 2 micrograms/ml, which is approximately 5% of the prekallikrein concentration in normal plasma. It appears that an acid-induced conformational change of the prorenin molecule is not required for its activation by plasma kallikrein.
This study concerns a case of congenital homozygous deficiency in alpha 2-antiplasmin associated with a severe hemorrhagic diathesis. Heterozygous family members also show a mild bleeding tendency. The propositus is a 17-yr-old male born of white parents and showing a severe hemorrhagic diathesis characterized by spontaneous bleeding in the joints since his early childhood. He was originally suspected of having factor XIII deficiency but was found to have normal functions of the coagulation system and the platelets. Except for alpha 2-antiplasmin, all protease inhibitors showed normal plasma values. With the immediate plasmin inhibition test (synthetic substrate), only 2% of normal functional inhibition was detected, while no reaction with monospecific antisera for alpha 2-antiplasmin was observed. Inhibition of activator-induced fibrinolysis in vitro was reduced. No enhanced spontaneous in vitro fibrinolysis was detected nor were there signs of increased in vivo fibrinolysis during an asymptomatic period. During recovery from a hemorrhagic episode, signs of previous consumption of antithrombin III, alpha 2-macroglobulin, factor XIII, and inter-alpha-trypsin inhibitor were noted. After the diagnosis was made, treatment with tranexamic acid (4 daily doses of 1 g) was effective for about 2 yr. Among the 37 family members studied, a separate group of 16 individuals (including the father and mother of the propositus) with approximately one-half normal plasma levels of alpha 2-antiplasmin both functionally (59% +/- 6%) and immunologically 48% +/- 8%) was discovered. The defect appeared to be inherited as an autosomal recessive gene; no ancestral consanguinity could be shown. The group of apparent heterozygotes as a whole showed increased levels of alpha 1-antitrypsin (142% +/- 39%; p less than 0.01), indicating systemic consequences of the deficiency and reduced binding (+/- 50%) of alpha 2-antiplasmin to fibrin. Six exhibited a mild hemorrhagic diathesis for which no explanation was provided by routine screening of coagulation and platelet functions; also, within the group of heterozygotes, the occurrence of the bleeding tendency did not correlate with differences in residual alpha 2-antiplasmin levels and functions. It is concluded that not only the absence of alpha 2-antiplasmin but also a reduction in its plasma level to +/- 60% of normal may predispose to a hemorrhagic diathesis.
Histidine-rich glycoprotein (HRG), a 3.8 S-alpha 2-glycoprotein recently identified as a new fibrinolysis regulating protein, was determined by an electroimmunoassay in samples of plasma collected during one menstrual or hormone cycle in 15 young women and in 11 women using oral contraceptives with 30 micrograms ethinyl oestradiol and 150 micrograms levo-norgestrel. Distinct individual levels caused the within-group variations of plasma HRG to be larger than the individual variations. In the hormone group the plasma HRG levels were decreased to about 75% of the normal, while the fluctuations during the cycle were minor in both groups.
Bovine plasma yields fibrinolytically inactive euglobulin fractions, even when prepared in the presence of dextran sulphate. Addition of flufenamate to these solutions only occasionally elicits a slight activity. However, highly fibrinolytic solutions are produced when euglobulins precipitated in the presence of dextran sulphate are exposed to chloroform. Evidence indicates that a plasminogen activator is formed, which subsequently converts plasminogen present in the fractions to plasmin. Bovine euglobulin fractions contain an inhibitor which seems to be specifically directed towards urokinase and not to plasmin or to tissue plasminogen activator. Its inhibiting capacity is decreased after treatment with chloroform.
3 patients with Behçet's disease were studied for their fibrinolytic status during exacerbation of the disease accompanied with thrombotic complications. All 3 patients exhibited low euglobulin fibrinolytic activity. This, however, could be attributed to an artificially increased coprecipitation of inhibitors, i.e., C1-inactivator in the euglobulin fractions. This phenomenon correlated with and was possibly related to inflammatory reactions in the patients. The most severely diseased patient only showed an increased plasmin inhibition in the plasma and a decreased response to venous occlusion. The increased plasmin inhibition was due to a slow type of inhibition, which could not be attributed to known protease inhibitors in blood. Its physiological relevance to fibrinolysis is questionable. There was no evidence for relevant general deviations in the fibrinolytic system in Behçet's disease; only for the severe case may the decreased fibrinolytic potency revealed by venous occlusion have contributed to the extent of the thrombotic manifestations.
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Fibrinolytic studies in euglobulin fractions of Fletcher trait plasma (deficient in prekallikrein) revealed reduced activities as compared to normal plasma. A quantitative assay for total plasminogen activator plus proactivator in plasma showed that the amount in Fletcher trait patients is about half of normal (normal = +/- 100 blood activator units [BAU]/ml). Plasma kallikrein partially purified in a high and low molecular weight form exerted plasminogen activator activity amounting to 10-15 BAU/ml plasma. So, the absence of kallikrein in the deficient plasma cannot fully account for the reduction in activator activity. Additions of kallikrein preparations or normal plasma fractions resulted in additional activator activity in Fletcher trait plasma which was assessed at 30-40 BAU/ml. This activity was assumed to originate from a previously undescribed plasminogen proactivator whose activation is kallikrein- and factor XII-dependent. Fractionation experiments demonstrated the presence of two major activities and a minor activity caused by kallikrein in normal plasma. It is concluded that plasma kallikrein has two functions in the generation of factor XII-dependent fibrinolytic activity: one as a direct plasminogen activator and another as a factor in the activation of a major factor XII-dependent plasminogen proactivator.
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A method for the assay of human plasma prekallikrein in which a chromogenic synthetic tripeptide, PPAN, is used as a substrate for kallikrein is described. The conversion of prekallikrein to kallikrein is achieved by cold activation (0 degrees C) with water-soluble dextran sulfate. Conditions for obtaining optimal amounts of free kallikrein with respect to concentration of dextran sulfate, activation time, inhibitors (C-1-inactivator), and requirement of factor XII have been determined. The activation procedure is compared to other known procedures. The assay system was worked out for pooled normal plasma and is applicable to any plasma sample not liable to unwanted preactivation or incomplete inactivation, as revealed by control experiments. A survey in 15 apparently health individuals showed a mean activity of 476 +/- 58 (S.D.) mU/ml with a range of 385 to 586 mU/ml.
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The fibrinolytic activity of euglobulin fractions prepared from human morning plasma and assayed on fibrin plates is strongly inhibited by the C1-inactivator present in the fractions. Flufenamate, a potent representative of the group of synthetic thrombolytic agents, eliminates this inhibition in euglobulin fractions. This elimination is an apparently irreversible reaction dependent on concentration, time and temperature. The fibrinolytic enhancing effect of flufenamate in euglobulin fractions correlated well with a similar effect of added C1s, which neutralized the C1-inactivator. The effect of flufenamate was slightly greater than that of added C1s, suggesting an additional effect of the flufenamate. The activity enhancing effect of the flufenamate at the lower molarities could be separated from an activity decreasing effect at the higher molarities. A simple technique by which inhibitory effects in euglobulin fibrinolysis are selectively eliminated is described.
Considerable amounts of C1 inactivator and inter-alpha-trypsin inhibitor pecipitate during euglobulin fractionation of human plasma. The amount precipitated depends on the ionic strength and the pH during the fractionation procedure. In contrast, alpha1-anti-trypsin, alpha2-macroglobulinand antithrombin II are present in euglobulin fractions in trace amounts only. The fibrinolytic activity of the euglobulin fractions is inhibited by the endogenous C1 inactivator, particularly as shown by comparison of normal and hereditary angioneurotic edema (HANE) plasma.
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