Phenotype-genotype correlation in eight Polish patients with inherited Factor XIII deficiency: identification of three novel mutations.

Inherited factor XIII (FXIII) deficiency is known as one of the most rare blood coagulation disorder in humans. In the present study, phenotype and genotype of eight FXIII deficient Polish patients from five unrelated families were compared. The patients presented with a severe phenotype demonstrated by a high incidence of intracerebral haemorrhages (seven of eight patients), haemarthrosis (six patients) and bleeding due to trauma (five patients). Introduction of regular substitution with FXIII concentrate prevented spontaneous bleeding in seven patients. In all patients, mutations within the F13A gene have been identified revealing four missense mutations (Arg77Cys, Arg260Cys, Ala378Pro, Gly420Ser), one nonsense mutation (Arg661X), one splice site mutation (IVS5-1 G>A) and one small deletion (c.499-512del). One homozygous large deletion involving exon 15 was detected by failure of PCR product. The corresponding mutations resulted in severely reduced FXIII activity and FXIII A-subunit antigen concentration, while FXIII B-subunit antigen remained normal or mildly decreased. Structural analysis demonstrated that the novel Ala378Pro mutation may cause a disruption of the FXIII catalytic triad leading to a non-functional protein which presumably undergoes premature degradation. In conclusion, the severe phenotype with high incidence of intracranial bleeding and haemarthrosis was in accordance with laboratory findings on FXIII and with severe molecular defects of the F13A gene.

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.

[Prevalence of G20210A prothrombin gene mutation in Poland]. / Wystepowanie mutacji G20210A genu protrombiny w Polsce.

The G20210A mutation of the prothrombin (PT) gene has recently been identified as a risk factor for venous thromboembolism (VTE). This mutation was shown to be present mainly among Caucasian populations, with a higher frequency in southern than in northern Europe. The aim of our study was to determine the prevalence of the PT 20210A allele in the Polish general population and in patients with a history of venous thrombosis. The patient group comprised 323 subjects with VTE before the age of 45, recurrent VTE or thrombosis in an unusual site. The control group consisted of 399 healthy individuals. Heterozygosity for the PT 20210A allele was found in 21 (6.5%) patients and 7 (1.8%) controls. In 7 (33.3%) of the 21 heterozygous patients the PT 20210A allele was associated with the factor V Leiden mutation, in 1--with the homozygous C677T mutation of the methylenetetrahydrofolate reductase (MTHFR), and in 1--with lupus anticoagulant. Our results indicate that the presence of the 20210A allele is a mild risk factor for venous thrombosis if not associated with other thrombophilic defect (odds ratio 2.2; 95% CI: 0.8-5.5). The risk is greater in double heterozygous carriers of the PT 20210A allele and factor V Leiden mutation.

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.

Degradation of glycophorin A of human erythrocytes in patients with myelo- or lymphoproliferative disorders: possible role of neutrophil proteases.

We have previously reported that glycophorin A (GPA) of human erythrocytes (carrying blood group M and N determinants) was totally digested by incubation of erythrocytes with human neutrophil elastase (HNE) and cathepsin G (CathG). The membrane-bound GPA fragments fractionated by SDS-PAGE gave characteristic patterns of bands detected by immunoblotting with the monoclonal antibody PEP80. Erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo. Characteristic electrophoretic patterns of bands derived from a partial GPA digestion were observed and these patterns were different for both enzymes and different from those obtained after total GPA digestion. GPA was also partially digested by incubation of erythrocytes with granulocytes in the presence of Ca2+ and calcium ionophore and electrophoretic pattern of digestion products was similar to that obtained with low doses of HNE. No GPA digestion products were detected after treatment of erythrocytes with plasmin and kallikrein. Untreated erythrocytes of 21 patients with various myelo- or lymphoproliferative disorders were tested by SDS-PAGE of RBC membranes and immunoblotting with the anti-GPA PEP80 antibody. GPA degradation products, resembling those formed by a mild CathG treatment of control RBC, were detected in nine patients. GPA fragmentation was in some cases accompanied by a reduced expression of blood group MN determinants. No distinct relation was observed between the occurrence of GPA degradation in erythrocytes and increases in plasma concentrations of HNE-alpha1-proteinase inhibitor (alpha1-PI) complex considered to be an indication of a release of neutrophil proteinases in vivo. However, the results suggested that a partial GPA degradation in haematological proliferative disorders may occur due to limited proteolysis by neutrophil proteinases, most likely by CathG.

Prevalence of heparin cofactor II deficiency in patients with a history of venous thrombosis.

Heparin cofactor II (HC II) is a plasma glycoprotein which inhibits thrombin but not factor Xa and which requires heparin or other glycosaminoglycans for its activation. Although several pedigrees have been reported in which 50% decreases in plasma HC II were associated with venous or arterial thrombosis, the role of HC II deficiency in inherited thrombophilia remains unproved. The present study was performed to determine the prevalence of HC II deficiency among patients with a history of venous thrombosis. HC II antigen was measured by electroimmunoassay in 122 unrelated patients with first episode of deep vein thrombosis developed before the age of 45 and in 114 healthy volunteers. Of the controls, 1 had a low HC II concentration (37%), while in the remaining 113, levels ranged from 65 to 180% with the mean value of 98.6 +/- 20.6%. In thrombosis patients, the mean HC concentration was 99.9 +/- 28.0%: individual values ranged from 52 to 180%. Seven patients (5.7%) exhibited values beneath the lower limit of the normal range (65%). These results indicate that HC II deficiency is more prevalent among patients with venous thromboembolism than in healthy subjects.

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.

Degradation of human erythrocyte surface components by human neutrophil elastase and cathepsin G: preferential digestion of glycophorins.

Human erythrocytes treated with purified human neutrophil elastase (HNE) or cathepsin G (CathG) were analysed by serological methods and by SDS-polyacrylamide gel electrophoresis followed by staining or immunoblotting with monoclonal antibodies. Both enzymes digested exhaustively glycophorins A, B and C, and HNE caused a partial digestion of band 3 protein. The degradation of other membrane proteins was not detectable by the methods used. Immunoblotting with the use of monoclonal antibodies against the defined epitopes of glycophorin A showed that HNE and CathG hydrolysed distinct peptide bonds in this antigen. The antibody PEP80, specific for the epitope in the cytoplasmic fragment of glycophorin A, gave patterns of bands which were characteristic for each of the two proteases. These bands could be distinctly identified in erythrocyte membrane samples containing only few percent of digested glycophorins. Therefore, the immunoblotting with this antibody may be useful as a sensitive assay for detecting the action of neutrophil proteases on red blood cells.

[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.

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.

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.

Cryoprecipitate of intermediate purity produced in a closed thaw-siphon system from DDAVP stimulated blood donor plasma.

The effects of intravenous administration of DDAVP to blood donors and the use of DDAVP plasma for the production of cryoprecipitate in the closed thaw-siphon system were evaluated. DDAVP treatment produced on the average a 3.2-fold rise in plasma levels of factor VIII. Von Willebrand factor antigen increased to a lesser extent. Cryoprecipitate prepared from 220-280 ml aliquots of DDAVP stimulated donor plasma contained 472 +/- 210 units of factor VIII and 276 +/- 130 units of von Willebrand factor antigen. The average yield of factor VIII was 57% of that in the prefrozen plasma. The specific activity of factor VIII in cryoprecipitate was 0.77 +/- 0.44 U/mg protein, comparable to that for intermediate purity concentrates. Thus, by the use of DDAVP and the thaw-siphon technique it is possible to produce cryoprecipitate 4-7 times as potent as conventionally manufactured preparations.

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.

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.

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.