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Biomedical subjects

M Furlan

Publications and source records attributed to M Furlan.

At least 19 recordsLinked to original sources
PubMed1997

Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura.

In patients with thrombotic thrombocytopenic purpura (TTP), excessive intravascular platelet aggregation has been associated with appearance in plasma of unusually large von Willebrand factor (vWF) multimers. These extremely adhesive vWF multimers may arise due to deficiency of a "depolymerase" cleaving vWF to smaller molecular forms, either by reducing the interdimeric disulfide bridges or by proteolytic degradation. We studied the activity of a recently described vWF-cleaving protease in four patients with chronic relapsing TTP. Diluted plasma samples of TTP patients were incubated with purified normal human vWF in the presence of a serine protease inhibitor, at low ionic strength, and in the presence of urea and barium ions. The extent of vWF degradation was assayed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. Four patients, that included two brothers, with chronic relapsing TTP displayed either substantially reduced levels or a complete absence of vWF-cleaving protease activity. In none of these patient plasmas was an inhibitor of or an antibody against the vWF-cleaving protease established. Our data suggest that the unusually large vWF multimers found in TTP patients may be caused by deficient vWF-cleaving protease activity. Deficiency of this protease may be inherited in an autosomal recessive manner and seems to predispose to chronic relapsing TTP. The assay of the vWF-cleaving protease activity may be used as a sensitive diagnostic tool for identification of subjects with a latent TTP tendency.

ADAM Proteins↗
PubMed1997

[The significance of APC resistance (activated protein C) for clinical practice].

Resistance against the anticoagulatory effect of activated protein C (APC resistance) is the most prevalent hereditary risk factor for venous thromboembolism known today. In this short review, an exemplary casuistic observation from our thrombophilia outpatient ward is presented. The discovery by Dahlbäck et al. in 1993 of hereditary APC resistance as a basis of familial thrombophilia and the molecular characterization of this defect as point mutation in the coagulation factor V gene (FV R506Q- or FV Leiden-mutation) by Bertina and coworkers are discussed. The clinical implications of this thrombophilia and relevant knowledge for daily practice are briefly summarized.

Adult↗
PubMed1997

Plasma protein C inhibitor is elevated in survivors of myocardial infarction.

Many studies have shown alterations of the hemostatic and fibrinolytic systems in patients with atherosclerotic disease, principally in levels of plasminogen activator inhibitor-1. However, in a large prospective study only fibrinogen, von Willebrand factor antigen, and tissue plasminogen activator antigen were found to be independent risk markers for acute coronary events. The present study evaluated the fibrinolytic system in coronary artery disease, paying particular attention to another inhibitor of fibrinolysis, plasminogen activator inhibitor-3, also called protein C inhibitor (PCI). One hundred fifteen nonanticoagulated male survivors of myocardial infarction were investigated for a range of hemostatic and fibrinolytic parameters that were compared with values in 87 age-matched healthy control male subjects. PCI active antigen was significantly (P < .03) elevated in the myocardial infarction group compared with the control group and was associated with the number of acute coronary events suffered (P = .005) but not with the severity of disease as determined by coronary angiography. Elevated PCI plasma levels can be considered as a risk marker for acute coronary events and might be of particular importance in the pathogenesis of this disease due to the interference of PCI in both the anticoagulant and fibrinolytic systems.

Adult↗
PubMed1997

von Willebrand factor without the A2 domain is resistant to proteolysis.

von Willebrand factor (vWF) is a complex multimeric plasma glycoprotein, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for factor VIII. vWF has a domain structure consisting of repeated A, B, C, and D domains. The A1 domain is involved in binding to the platelet receptor glycoprotein (GP) Ib, and the A3 domain has a binding site for collagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A patients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, delta A2-vWF. Previous studies have shown that this approach is a powerful tool to study the function of a domain in a protein since it does not affect the activity of other domains. After expression in baby hamster kidney (BHK) cells, delta A2-vWF was compared to wild-type (WT) vWF, and to delta A1-vWF (Lankhof et al., Blood 86: 1035, 1995). Ristocetin induced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen type III. Adhesion studies to surface coated purified delta A2-vWF or to delta A2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that delta A2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfolding of the molecule is necessary for exposure of the cleavage site. delta A2-vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.

Animals↗
PubMed1996

Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis.

Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximately 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximately 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. vWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, of calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N-ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe-CHN2. Activation by divalent metal ions was found to increase in the following order: Zn2+ approximately Cu2+ approximately CD2+ approximately Ni2+ approximately Co2+ <Mn2+ <Mg2+ <Ca2+ <Sr2+ <Ba2+. The observed properties of the vWF-degrading enzyme differ from those of all other hitherto described proteases. Purified vWF was incubated with the protease, and the degraded material subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis after disulfide reduction. The size, amino acid composition, and amino terminal sequence of the reduced fragments confirmed that the peptide bond 842Tyr-843Met had been cleaved, ie, the same bond that has been proposed to be cleaved in vivo.

ADAM Proteins↗
PubMed1996

Spontaneous neurological recovery after stroke and the fate of the ischemic penumbra.

We prospectively tested the hypothesis that early recovery after ischemic stroke depends on the ultimate survival of functionally impaired, critically ischemic (i.e., "penumbral") tissue. From a series of 26 consecutive patients studied with positron emission tomography within 18 hours of first-ever stroke in the middle cerebral artery territory, all 11 survivors to the 2-month end point who exhibited increased oxygen extraction fraction were declared eligible. The positron emission tomographic images were compared to ultimate infarction defined by computed tomography performed during the chronic stage. The penumbra (operationally defined by increased oxygen extraction fraction and divided outcome despite uniformly reduced cerebral blood flow) was individually detected in 10 of the 11 patients; cerebral blood flow ranged from 7 to 17 ml/100 gm x min, consistent with that found in monkey studies. The volume of the penumbra that escaped infarction was highly correlated with neurological recovery (p < 0.04 to p < 0.0001, depending on the scale used). This longitudinal study is the first to characterize the penumbra in humans and to document one mechanism strongly influencing recovery; the surviving penumbra may offer opportunities for secondary perifocal neuronal reorganization. Therapeutic measures to prevent infarction of the penumbra (up to 16 hours in this series) may have reduced residual neurological impairment. Mapping the extent of the penumbra, according to prospective criteria, may allow one to predict each patient's potential for recovery, and to select the most appropriate candidates for therapeutic trials.

Aged↗
PubMed1996

Von Willebrand factor: molecular size and functional activity.

Von Willebrand factor (vWF) is the largest protein found in plasma. It circulates in blood as a series of multimers ranging in size from 500 to 20,000 kDa. The variable molecular weight of vWF is due to differences in the number of subunits comprising the protein. vWF mediates platelet adhesion to subendothelium of the damaged blood vessel. Only the largest multimers are hemostatically active. Each vWF subunit contains binding sites for collagen and for platelet glycoproteins GPIb and GPIIb/IIIa. Multiple interactions of repeating binding sites in vWF multimers with adhesive protein(s) of the subendothelium and with receptors on the platelet surface lead to "irreversible" binding of platelets to the exposed subendothelium. Functional properties of vWF are typical of multisubunit proteins encoded by autosomal loci. The phenotype of von Willebrand disease is determined by the properties of the dysfunctional subunits which become incorporated into heteropolymeric forms of vWF. Absence of large vWF multimers, seen in type 2A von Willebrand disease and in myeloproliferative disorders, is associated with bleeding tendency. On the other hand, in patients with vWF multimers of supranormal size, as they occur in thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), there is an increased risk of thrombosis. Proteolytic enzyme(s) are involved in physiologic regulation of the polymeric size of vWF. We have purified from human plasma a protease cleaving vWF at the same peptide bond that is also cleaved in vivo. vWF was quite resistant against the protease in a physiologic buffer but was degraded at low salt concentration or in the presence of 1 M urea. It appears that a conformational change in the vWF molecule exposes the specific protease-sensitive peptide bond and thus enhances degradation of vWF multimers. In some variants of type 2A vWF, the cleavage site in the vWF subunit is more susceptible to proteolytic degradation than in normal vWF, whereas in patients with TTP or HUS the protease activity may be suppressed. vWF-degrading protease plays an important role in pathogenesis of congenital or acquired disorders of hemostasis and thrombosis.

Blood Platelets↗
PubMed1996

Early spontaneous hyperperfusion after stroke. A marker of favourable tissue outcome?

To clarify the relationships between early hyperperfusion (i.e. the hallmark of early, efficient recanalization in animal stroke models) and ultimate infarction, we have compared acute-stage perfusion PET images and chronic-stage CT scans in patients with middle cerebral artery (MCA) stroke. We used PET and the oxygen-15 (15O) equilibrium method to obtain cerebral blood flow (CBF), cerebral blood volume (CBV), oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen consumption (CMRO2) parametric images in 30 consecutive, still symptomatic, first-ever MCA territory stroke patients without sign of haemorrhage at admission CT scan. Each subject was studied twice, first within 5-18 h of stroke onset, and, in survivors, approximately 1 month later; a plain CT scan (co-registered with PET) was performed approximately 1 month after onset. Following initial screening based on acute-stage perfusion images, 10 survivors with focal hyperperfusion in the appropriate MCA territory confirmed by computer were declared eligible. In each patient, the topography and volume of both hyperperfusion and infarction (delineated on late CT scan) were recorded, and all PET parameters were obtained for both areas and both times. The hyperperfused areas affected the cortical MCA territory, often widely so and in a patchy fashion; they were topographically distinct from, and consistently larger than (P < 0.01, Wilcoxon sign test) the final infarcts, which were small and generally deep-seated. In none of the nine patients in whom it was successfully performed did transcranial Doppler reveal MCA stem occlusion. In the hyperperfused regions, the acute-stage perfusion, blood volume and oxygen consumption were significantly increased, and the OEF significantly reduced, while all these variables had significantly returned toward normality in the chronic-stage PET study. The ultimately infarcted area did not exhibit significant hyperperfusion in the acute stage. The areas with acute-stage hyperperfusion exhibited haemodynamic and metabolic abnormalities consistent with post-recanalization hyperperfusion, i.e. vasodilatation and "luxury perfusion'. Increased oxidative metabolism, previously reported only in animals, presumably reflects an overshoot of protein synthesis. The fact that the areas with hyperperfusion, though extensive, were topographically distinct from the infarcted region, suggests that spontaneous non-haemorrhagic hyperperfusion, when documented 5-18 h after onset, is a harmless and even perhaps beneficial phenomenon. These results have implications for clinical trials.

Aged↗
PubMed1996

Binding of calcium ions and their effect on clotting of fibrinogen Milano III, a variant with truncated A alpha-chains.

Calcium ions are known to be required for normal polymerisation of fibrin monomers. Normal human fibrinogen has three high-affinity calcium binding sites. Two of these are located in the D-domains whereas the third binding site was tentatively assigned either to the E-domain or to the C-terminal part of the A alpha-chain. Furthermore, binding of calcium to the low-affinity binding sites (n > or = 10) facilitates fibrin monomer polymerisation. In several abnormally clotting fibrinogen variants, the polymerisation defect was partially normalised following addition of calcium ions. In this study, we show normal binding of calcium to fibrinogen Milano III, a homozygous fibrinogen variant with truncated A alpha-chains (A alpha 452 Gly-Pro-Asp-->Trp-Ser-Stop). These results confirm that the C-terminal parts of the A alpha-chains beyond residue 451 Ile are not involved in calcium binding. The thrombin time was severely prolonged and the final clot turbidity was strongly reduced in fibrinogen Milano III. Moreover, calcium ions did not significantly improve the abnormal clotting behavior of this dysfibrinogen. The polymerisation defect in fibrinogen Milano III appears to be due to truncated A alpha-chains as well as to the disulphide-linked albumin.

Blood Coagulation↗
PubMed1996

Normal binding of calcium to five fibrinogen variants with mutations in the carboxy terminal part of the gamma-chain.

Calcium ions are known to accelerate polymerization of fibrin monomers. Each of the two carboxy terminal domains of normal fibrinogen contains one high-affinity calcium binding site that seems to be situated close to the polymerization site in the gamma-chain. Most hitherto described functionally defective fibrinogen variants showed impaired clot formation. Since the tightly bound calcium ions may influence the conformation of the polymerization site, the question arises whether the abnormal clotting of a dysfibrinogen might be due to defective calcium binding. We investigated binding of calcium to fibrinogen and the effect of calcium on the clotting properties of five heterozygous fibrinogen variants showing normal thrombin-induced fibrinopeptide release but abnormal polymerization of fibrin monomers. Each of these dysfibrinogens has one single amino acid substitution in the carboxy-terminal part of the gamma-chain: fibrinogen Claro (gamma 275 Arg-->His), Milano V (gamma 275 Arg-->Cys), Milano I (gamma 330 Asp-->Val), Bern I (gamma 337 Asn-->Lys), and Milano VII (gamma 358 Ser-->Cys). The shortest thrombin clotting time and the earliest onset of turbidity increase were observed in fibrinogen gamma 358 Ser-->Cys; both parameters were little affected by calcium concentration. In the variant gamma 337 Asn-->Lys, the thrombin time was abnormally prolonged at 0.01 mM Ca2+, but it was normalized at 1 mM calcium. In contrast, the abnormal fibrin polymerization of fibrinogen gamma 330 Asp-->Val was barely improved at increasing calcium concentrations. Both variants with the substitution of gamma 275 Arg, the residue indispensable for normal D:D interactions, showed the slowest rate of fibrin polymerization and the lowest turbidity of fibrin clots at any Ca2+ concentration used. High affinity calcium binding was found to be normal in all five fibrinogen variants studied, suggesting that their abnormal clotting was not due to defective binding of calcium. The gamma-chain in the fragment D1 derived from the variant gamma 337 Asn-->Lys was further degraded by plasmin in the presence and in the absence of calcium, whereas fragments D1 from the other four gamma-chain variants as well as from normal fibrinogen were protected against plasmic degradation in the presence of 1 mM Ca2+.

Amino Acid Sequence↗
PubMed1995

Improved detection of proteolytically cleaved high molecular weight kininogen by immunoblotting using an antiserum against its reduced 47 kDa light chain.

Several ligand blotting or immunoblotting assays for the detection of single-chain and proteolytically cleaved two-chain high molecular weight kininogen (HK) in whole plasma have been described. Since they may suffer from poor sensitivity for the light chain species of cleaved HK on reduced blots, an antiserum against the reduced and alkylated 47 kDa light chain of HK was raised in rabbits allowing improved immunodetection of HK species on blots of reduced electropherograms. This immunoblotting method is highly specific and sensitive, permitting detection of 0.2 ng single-chain HK or the light chains of 2 ng proteolytically cleaved HK in whole plasma. Thus, this immunoblotting technique is at least 50-100 times more sensitive than ligand blotting with radiolabelled factor XI overlay. A similar cleavage pattern was observed following in vitro activation of normal human plasma by dextran sulphate and after plasma kallikrein-induced proteolysis of purified HK. However, bands of different molecular weights were generated after HK had been cleaved by purified leukocyte elastase. During acute attack in a patient with hereditary angioedema, high levels of in vivo cleaved HK were noticed, whereas concentration of cleaved HK in plasma samples and synovial fluids from patients suffering from various inflammatory conditions were not substantially higher than those in normal plasma. During in vitro cold activation of plasma samples of pregnant women concomitant HK cleavage and plasma kallikrein generation were observed.

Angioedema↗
PubMed1995

No association of APC resistance with myocardial infarction.

Resistance to activated protein C (APC resistance) due to the factor V mutation 506 Arg-->Gln (factor V Leiden) is the most prevalent inherited risk factor for venous thromboembolism. Its association with arterial thromboembolic disease, however, is still controversial. In the present study we found no difference between the prevalence of APC resistance (assessed by the ratio of the aPTT with and without added APC) in 134 non-anticoagulated survivors of myocardial infarction and that in 100 controls of similar age and sex distribution (2.2% and 2.0%, respectively). Patients showed a significantly higher median value for the aPTT ratio than controls (2.85 and 2.66, respectively), a fact we could not explain by our data.

Adult↗
PubMed1994

A frameshift mutation in Exon V of the A alpha-chain gene leading to truncated A alpha-chains in the homozygous dysfibrinogen Milano III.

An inherited dysfunctional fibrinogen variant, denoted as fibrinogen Milano III, was found in a 13-year-old girl suffering from recurrent venous thrombosis. Plasma of the patient exhibited prolonged thrombin time and Reptilase time. Polymerization of fibrin monomers in the presence and absence of calcium ions was strongly impaired. SDS-polyacrylamide gel electrophoresis of reduced fibrinogen showed normal B beta- and gamma-chains, whereas no normal A alpha-chain was detected in the proposita. Immunoblot analysis with the monoclonal antibody Y18, detecting an epitope within the stretch of amino acids A alpha 1-51, revealed an A alpha-chain of about 50 kDa with an intact amino terminus. Immunoblotting with antibodies directed against serum albumin demonstrated the presence of albumin covalently linked to fibrinogen via a disulfide bridge. The structural defect of fibrinogen Milano III was determined by sequence analysis of a single-stranded fragment of genomic DNA amplified by polymerase chain reaction. An insertion of a thymine in the exon V of the A alpha-chain gene after the triplet ATT coding for IleA alpha 451 altered the reading frame and caused premature termination of the protein synthesis (Trp452(TGG)-Ser453(TCC)-Stop454(TGA)). In both parents, normal and mutant alleles were established, leading to duplication of the sequence pattern after the thymine insertion site, whereas the proposita is homozygous for the new mutation in the fibrinogen A alpha-chain gene.

Base Sequence↗
PubMed1994

A new substitution, gamma 358 Ser-->Cys, in fibrinogen Milano VII causes defective fibrin polymerization.

Fibrinogen Milano VII is a hereditary fibrinogen variant detected in a woman with no clinical symptoms of bleeding or thrombosis. Thrombin and reptilase clotting times were prolonged in six family members from three generations. Release of fibrinopeptides A and B was normal. Fibrin polymerization was strongly delayed both in the presence and in the absence of calcium. The structural defect was determined by sequence analysis of a 290-bp fragment of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet TCT coding for the amino acid residue gamma 358 was found to be replaced by TGT, resulting in the substitution gamma 358 Ser-->Cys. Immunoblot analysis demonstrated the presence of covalently linked fibrinogen albumin and fibrinogen (albumin)2 complexes. Albumin was released from fibrinogen Milano VII by limited reduction with 2-mercaptoethanol. Fibrin polymerization was not normalized after removal of albumin from fibrinogen Milano VII, suggesting that the delayed clot formation is not due to steric hindrance caused by bound albumin but by substitution of gamma 358 Ser by Cys itself. Our results indicate that the residue gamma 358 Ser is essential for normal expression of the carboxy terminal polymerization site on the fibrinogen gamma-chain.

Adult↗