Difference between type I autoimmune inhibitors of fibrin stabilization in two patients with severe hemorrhagic disorder.

Inhibitors of fibrin stabilization of apparently autoimmune origin, found in two severely bleeding unrelated patients (W. G. and G. A.), were compared with regard to their biological target specificities, potencies and immunological characteristics. Both interfered only with the activation of fibrin stabilizing factor (coagulation Factor XIII) and, while totally preventing the conversion of this zymogen to the functional transamidating enzyme, fibrinoligase (Factor XIII(a)), they showed very little inhibition toward the enzyme itself. Thus, according to the classification of Lorand concerning biological specificities, both can be characterized as Type I inhibitors of fibrin stabilization. Potencies of the two inhibitors were quite similar when measured in conjunction with the plasma zymogen, but they differed remarkably in tests with platelet Factor 13. The inhibitor of patient W. G. prevented the activation of the zymogen from platelets, but that of G. A. had no effect on the platelet factor. It may therefore be concluded that the inhibitor of W. G. is directed exclusively against the a subunit which is a common constituent of plasma as well as platelet factors. The inhibitor of G. A., however, must be targeted against determinants uniquely characteristic for the ab ensemble of the plasma zymogen including the b subunit. On the basis of this difference in target specificity, the inhibitor of W. G. is designated as Type I-1 and that of G. A. as Type I-2. The inhibitors of both patients were isolated as immunoglobulins, and neutralization tests revealed that the antibody of W. G. comprised mainly heavy chains of the IgG1 and light chains of the kappa class. The antibody of G. A. proved to be considerably more heterogeneous and contained IgG1 and IgG3 heavy chains as well as kappa- and lambda-light chains. The finding that the antibody of W. G. inhibited conversion of platelet Factor 13 and also its thrombinmodified form, but had no effect on the thrombin and Ca(2+)-activated factor, is an indication that antigenic determinants existing both on the native zymogen and on its hydrolytically modified form become buried in the Ca(2+)-dependent step of activation. This is clear evidence for the occurrence of a significant conformational change in the protein structure attendant to the process of unmasking of its enzymic activity.

Characterization of a plasminogen activator secreted by cultured bovine aortic endothelial cells.

A plasminogen activator was purified from the serum-free conditioned medium of bovine aortic endothelial cell cultures by chromatography on zinc chelate-agarose and benzamidine-CH-Sepharose. The final material consisted of a main fibrinolytically active component with Mr 30,000 and a minor component with Mr 41,000. It was obtained with a yield of 60%, a purification factor of 35 and a purity of 25-50%. The activity of this plasminogen activator was completely neutralized by antibodies to human urokinase but not by antibodies against human tissue plasminogen activator. Purified tissue plasminogen activator from bovine heart, however, was completely neutralized by antibodies against human tissue plasminogen activator but unaffected by antibodies to human urokinase. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like plasminogen activator, and not a tissue-type plasminogen activator as was generally assumed.

Human platelets release alpha-6-L-fucosyltransferase upon activation.

Previously we have shown that human platelets release alpha-6-L-fucosyltransferase (EC 2.4.1.68) during coagulation of blood [(1987) Glycoconjugate J. 4, 43-49]. Here we report that agonists which induce platelet aggregation bring about release of the enzyme. In quantitative terms the release of alpha-6-L-fucosyltransferase by washed, aggregated platelets was very similar to that occurring during blood coagulation.

Effect of proteolysis on quantitation of plasma fibronectin concentration by two immunoassays (electroimmunoassay and immunoturbidimetric technique).

Quantitation of fibronectin (FN) concentration is strongly influenced by FN fragmentation with trypsin, kallikrein and plasmin. Digestion by trypsin and kallikrein leads to a progressive decline in FN detectability by the immunoturbidimetric technique to zero values but is associated with an increase in the height of rockets in the Laurell's electroimmunoassay. Plasmin mediated FN fragmentation induces a strong overestimation of the FN content by the electroimmunoassay and, at very high enzyme concentrations, provokes an underestimation of FN by the immunoturbidimetric technique. The decline in FN reactivity in the immunoturbidimetric assay coincides with the disappearance of heavy fractions migrating only slightly faster than native FN in SDS-PAGE. The increase in the height of rockets in the electroimmunoassay is the highest when fractions of intermediate rate of migration prevail in the SDS-PAGE pattern. Concomitant use of these two immunoassays can distinguish native FN from its degraded form and may possibly provide a partial explanation for discrepancies in published studies on the concentration of circulating FN in various pathological states.

Purification and characterization of the plasminogen activator secreted by a rat brain tumor cell line in culture.

The plasminogen activator secreted by a cultured rat brain tumor cell line (RT4-71-1) (Imada M. and Sueoka N., Develop. Biol. 69, 97-107, 1978) was purified by chromatography on zinc chelate-agarose, concanavalin A-agarose and Sephadex G-150 in the presence of 0.01% (vol/vol) Tween 80. Aprotinin was added to the culture medium to a concentration of 20 KIU per ml and to the buffers in the first two chromatographic steps to a concentration of 10 KIU per ml. Approximately 90 microgram purified material was obtained from 11 of culture medium with a yield of 39% and a purification factor of 200. Sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence of reducing agents showed one main band with Mr of about 60,000, and a minor band with Mr about 30,000. Fibrinolytic activity was associated with the main band. The rat brain tumor plasminogen activator bound to a fibrin clot to a similar extent as human tissue plasminogen activator, whereas urokinase did not bind. In quenching experiments of the fibrinolytic activities the purified rat brain tumor plasminogen activator appeared to be immunologically related to the human tissue plasminogen activator but unrelated to urokinase.

Different effects of human neutrophil elastase on platelet glycoproteins IIb and IIIa of resting and stimulated platelets.

The effect of human neutrophil elastase (HNE) on the structure and receptor activity of platelet glycoprotein IIb/IIIa complex was studied. Resting platelets, which bound only traces of 125I-fibrinogen in the absence of ADP, were found to be barely susceptible to HNE. As shown by immunoblotting experiments, treatment of such platelets with HNE (14 micrograms/ml) did not provoke a detectable cleavage of GPIIb but resulted in a partial digestion of GPIIIa and appearance of 110 kDa fragment. Such proteolytic modification of the GPIIb/IIIa complex was accompanied by a slight increase in the binding of fibrinogen to blood platelets in the absence of ADP. Treatment of partially activated platelets (spontaneous activation during washing procedure) with HNE caused a progressive loss of GPIIb and degradation of GPIIIa to 110 kDa and 60 kDa fragments. These spontaneously stimulated platelets had initially a high number of fibrinogen binding sites exposed, corresponding to approximately 50% of receptor capacity observed in platelets activated by the optimal concentration of ADP. Digestion of GPIIb/IIIa by HNE of such platelets markedly increased the exposure of fibrinogen receptors. Thus, the stimulation of platelets increases significantly the susceptibility of the GPIIb/IIIa complex to proteolysis by HNE. However, such modification of the GPIIb/IIIa does not destroy its function as a receptor for fibrinogen either on the resting or activated platelets.

Venostasis but not DDAVP infusion provokes the plasma fibronectin increase.

Plasma fibronectin (pFN), von Willebrand factor antigen (vWf:Ag), factor VIII procoagulant activity, fibrinogen, euglobulin lysis time (ELT) and hematocrit were determined in healthy blood donors before and after venostasis as well as after intravenous infusion of 1-deamino-8-D-arginine vasopressin (DDAVP). Both venostasis and DDAVP provoked an increase in vWf:Ag and shortening in the ELT. In contrast, venostasis only but not DDAVP induced an increase in pFN levels which was statistically significant with and without correction for a concomitant hematocrit increment. The results indicate that there is a distinct difference in the patterns of venostasis and DDAVP mediated release of proteins from the vessel wall.

Effect of neutral proteases from blood leukocytes on human platelets.

Two highly purified neutral proteases from human leukocytes i.e. elastase-like protease (ELP) and chymotrypsin-like protease (CLP) do not destroy human platelets since no difference was found in 51Cr liberation from control and enzyme-treated platelets. As with pancreatic chymotrypsin (alpha-CT) ELP does not induce the release of 3H-serotonin while CLP provokes 3H-serotonin secretion, in an enzyme concentration and time dependent fashion. The rate and degree of 3H-serotonin release by CLP is similar to that produced by thrombin. Incubation of platelets at 37 degrees C for 30 min with alpha-CT or ELP renders them resistant to thrombin-releasing activity. Thrombin did not liberate any additional label from platelets which lost over 60% of serotonin during the preceding incubation with CLP. alpha-CT and ELP do not aggregate platelets either in the presence or absence of apyrase. CLP does aggregate platelets suspended in Tyrode buffer without apyrase but not in the presence of apyrase (100 mg/l). The action of alpha-CT, ELP and CLP on washed platelets induces a progressive prolongation of lag phase and a decrease in changes of light transmission during aggregation by thrombin. Similarly to alpha-CT-treated platelets, those subjected to CLP action aggregate in the presence of human fibrinogen. It is concluded that: (1) neutral proteases possibly contribute to development of defects in platelet function in pathological states associated with liberation of leukocyte content into the circulation, (2) CLP similarly to alpha-CT, exposes fibrinogen receptors but in contrast to alpha-CT, CLP aggregates platelets and stimulates serotonin secretion.

Anticoagulant properties of extracellular slime substance produced by Staphylococcus epidermidis.

Slime produced by S. epidermidis strain KH 11 was extracted with phenol-saline. The saline phase was fractionated on a DEAE-Sepharose CL-6B column. The crude slime solution and its phenol-saline fraction were found to possess anticoagulant properties. They inhibited the coagulation of human plasma by thrombin, prolonged the activated partial thromboplastin time, but did not change the rate of plasma coagulation by reptilase. The anticoagulant effect of slime could be neutralized by rather high concentrations of protamine sulphate. In the presence of plasma, the staphylococcal slime also inhibited in a concentration dependent fashion the amidolytic activity of thrombin and factor Xa against synthetic chromogenic substrates. Both antithrombin III (AT III) and other plasma component(s), presumably heparin cofactor II, were required for the full expression of the slime effect. The anticoagulant action of slime was markedly less AT III dependent than that of heparin. The activity was resistant to heating (100 degrees C, 30 min). Slime and its fractions were stronger inhibitors of thrombin than of factor Xa. Fraction IV separated by DEAE-Sepharose chromatography and particularly rich in galactose and glucuronic acid had the highest inhibitory potency. It is conceivable that slime component(s) similar to glycosaminoglycans from other sources can carry the anticoagulant activity.

Effects of neutral proteases from human leukocytes on structure and biological properties of human factor VIII.

Human factor VIII was purified from cryoprecipitate and incubated for up to 24 hours with four neutral proteases of human blood leukocytes, namely, with elastase-like protease (ELP), chymotrypsin-like protease (CLP), collagenase and gelatinase. Electrophoretic patterns showed a reproducible sequence of degradation of factor VIII and of its 230,000 molecular weight subunit by ELP and CLP. Intermediate products were similar but those resulting from exhaustive proteolysis by ELP and CLP differed distinctly from each other. Procoagulant activity of factor VIII was rapidly and completely destroyed by ELP and CLP before visible electrophoretic changes would be detected. No increase in this activity was observed prior to its destruction. Von Willebrand factor (ristocetin cofactor) activity was considerably more resistant to ELP and CLP and declined in rough relation to degradation of highly aggregated forms of factor VIII. ELP and CLP produced a pronounced progressive increase in the Laurell reaction antigen. Normal human plasma showed a high potency to inhibit ELP and CLP. Large doses of these enzymes (300 microgram per ml) produced in the plasma medium only a moderate fall in factor VIII procoagulant activity. Collagenase and gelatinase did neither degrade factor VIII nor change its biological properties.

Alterations of blood platelet function induced by neutral proteases from human leukocytes.

Washed human platelets are damaged by two neutral proteases from human leukocytes (elastase-like protease, ELP and chymotrypsin-like protease, CLP). The damage is manifested as inhibited aggregation by ristocetin and collagen, and enhanced aggregation by ADP in the presence of fibrinogen. Similarly to alpha-chymotrypsin (alpha-CT), CLP also increases binding of 125I-fibrinogen to platelets and renders platelets aggregable by human fibrinogen. ELP is less effective in this respect possibly due to damage to platelet receptors for fibrinogen. In the plasma medium platelets are not sensitive to leukocytic proteases added at concentrations that provoke some prolongation of the time of plasma clotting by thrombin.

Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene.

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.

Factor V Leiden, prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase C677T genotype in young adults with ischemic stroke.

Ischemic stroke in young adults is a well-known disease, but despite extensive clinical and laboratory investigations, its etiology remains unclear in approximately half of the cases. We examined the prevalence of factor V Leiden, the prothrombin G20210A genotype, and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in 100 patients (51 males and 49 females) who survived an ischemic stroke without a cardiac embolic source at an age < or = 45 years, and in 238 healthy control subjects from the same geographic area. The patients were selected for study only if the diagnosis of stroke was documented by computed tomography scan or nuclear magnetic resonance (NMR) of the brain, or both. Heterozygosity for the FV Leiden mutation was found in 3 patients (3.0%) and in 10 control subjects (4.2%). Two patients (2.0%) and five control subjects (2.1%) were heterozygous for the prothrombin G20210A mutation. The frequencies of the MTHFR 677TT, CT, and CC genotypes in the patient group were 12%, 37%, and 51%, respectively, and were not significantly different from those in control subjects (11%, 40%, and 49%, respectively). In conclusion, our results indicate that FV Leiden mutation, prothrombin G20210A genotype, and homozygosity for the C677T mutation in the MTHFR gene are not associated with an increased risk for ischemic stroke in young adults.

[Quinidine-induced thrombocytopenia]. / Maloplytkowosc zalezna od chinidyny.

We present the case of drug-dependent thrombocytopenic purpura due to the treatment with quinidine. IgG quinidine-dependent antibodies in patient serum were detected in the platelet immunofluorescence test. They were bound to GP Ib on platelet membrane.

[Isolation and some properties of factor VIII (antihemophilic factor)]. / Izolowanie i niektóre wlasciwosci czynnika VIII (czynnika przeciwhemofilowego)

A method for isolation of factor VIII from cryoprecipitate fraction of human plasma has been elaborated. The isolation procedure involves precipitation with dextran, removal of fibrinogen by means of defibrase, precipitation with ammonium sulfate, polyethylene glycol fractionation, and Sepharose 6B gel filtration step. Factor VIII has been purified 7000- to 13,000 -- fold.. The preparation is homogenous by ultracentrifugal examination and it has an S20,w value of 19.4. It also shows a single precipitin line when subjected to immunoelectrophoresis employing rabbit antibodies against factor VIII. The preparation did not enter a 7.5% polyacrylamide gel containing sodium dodecyl sulfate even in the presence of 8 M urea. After reduction of the protein with 2-mercaptoethanol, subunits were formed which migrated as one band in polyacrylamide gel electrophoresis.

[Detection of Hemophilia A carrier state]. / Wykrywanie stanu nosicielstwa hemofilii A.

Factor VIII biological activity and factor-VIII-related antigen were measured in the plasma of 78 definite, probable or potential carriers of haemophilia A and in a control group of 74 normal women. Low values of factor VIII activity could be detected in 11 of 24 definite carriers, in 11 of 26 probable carriers and, in 10 of 28 potential carriers. On the other hand, the ratio of factor VIII activity to factor-VIII-related antigen was significantly lowered in 79 percent of definite carriers, in 50 percent of probable carriers and, in 21 percent of potential carriers. These studies indicate that determinations of both factor VIII activity and antigen is more reliable procedure in the detection of the carrier state for haemophilia A than determinations of factor VIII activity alone.

Effects of human neutrophil elastase and cathepsin G on the reactivity of platelets with antiplatelet antibodies.

Two human neutrophil serine proteases, elastase (HNE) and cathepsin G (CathG), are known to change the structure and hemostatic function of platelet surface membrane. The platelet membrane contains glycoproteins (GPs) which function as alloantigens, autoantigens and targets of drug-induced antibodies. The aim of this study was to investigate whether proteolysis of platelet GPs by HNE and CathG is associated with changes in the reactivity of platelets to antiplatelet antibodies. The platelet immunoreactivity was examined using the MAIPA (monoclonal antibody-specific immobilization of platelet antigens) assay and PSIFT (platelet suspension immunofluorescence test). The treatment of platelets with HNE led to a moderate increase in their reactivity to quinidine-dependent (anti-GP Ib) antibody and to a slight decline in the expression of HPA-1a. In contrast, CathG did not provoke any significant changes in platelet reactions with quinidine dependent and anti-HPA-1a antibodies. Both enzymes had no significant effect on the expression of HLA-A2, HLA-A3, HLA-B7 and HLA-B8 on platelets.

Secretory response of the vessel wall to DDAVP and venous occlusion in von Willebrand's disease.

The aim of this study was to compare the secretory response of the vascular wall in vivo to DDAVP (i.v. 0.3 microgram/kg, 30 min) and to venous occlusion (VO, 20 min) in control healthy subjects, patients with von Willebrand's disease type I (vWd I) and patients with von Willebrand's disease type III (vWd III). In controls (n = 10) and vWd I (n = 12), DDAVP induced a 2 to 3-fold rise in plasma von Willebrand factor antigen (vWf: Ag), factor VIII coagulant activity (VIII: C) and tissue--type plasminogen activator antigen (t-PA:Ag). VO was less effective in increasing vWf: Ag and VIII:C but produced a greater rise in t-PA:Ag. Large increments (over 10-fold) were observed in plasmin-alpha 2-antiplasmin complexes following both stimuli. In vWd III (n = 10), DDAVP and VO failed to increase vWf:Ag, VIII:C and t-PA:Ag. No significant changes in plasmin-alpha 2-antiplasmin complexes were observed in this group. Moreover, the baseline t-PA:Ag values were significantly lower in vWd III (2.17 +/- 1.13 ng/ml) than in controls (4.84 +/- 1.97 ng/ml, p < 0.001). A significant increase in urokinase--type plasminogen activator antigen (u-PA:Ag) was found only in controls after VO. Neither controls nor patients with vWd showed any changes in plasma fibronectin levels following DDAVP. The low t-PA:Ag results and the abnormal fibrinolytic response to DDAVP and VO in patients with severe (type III) vWd indicate that their endothelial cell abnormality is more extensive than the defect in the synthesis or release of vWf.