A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis.

Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner.

Anti-plasminogen antibodies compromise fibrinolysis and associate with renal histology in ANCA-associated vasculitis.

Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA.

ABO blood group genotypes, plasma von Willebrand factor levels and loading of von Willebrand factor with A and B antigens.

ABO blood group is a genetic determinant of von Willebrand factor (VWF) levels. We investigated the effect of ABO genotypes on VWF and factor VIII (FVIII) levels and on the degree to which VWF is loaded with A- and B-antigens, expressed as normalized ratios, nA-ratio and nB-ratio, respectively, in the Leiden Thrombophilia Study, a large case-control study on venous thrombosis. We found that the ABO locus had an allele-specific, dosage dependent effect on VWF and FVIII levels and on the loading of VWF with A-antigen and B-antigen.

von Willebrand factor and its propeptide: the influence of secretion and clearance on protein levels and the risk of venous thrombosis.

Background And Objectives: Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis.

Association between thrombin activatable fibrinolysis inhibitor genotype and levels in plasma: comparison of different assays.

Thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels exhibit a large interindividual variability in which genetic control seems to play a major role. However, recent reports have questioned the association between TAFI concentration and genotype, suggesting that variable antibody reactivity towards TAFI isoforms, particularly the Thr325Ile polymorphism (1040C/T), may lead to artefacts in TAFI antigen levels. In order to compare assay outcome we determined plasma TAFI levels in 92 healthy individuals, using an enzyme-linked immunosorbent assay (ELISA) (commercial antibodies), an electroimmunoassay (in-house antibodies) and a commercial chromogenic assay (Actichrome TAFI).

Alterations in the extrinsic pathway in hypertriglyceridemia do not cause a 'procoagulant state': effects of bezafibrate therapy.

Hypertriglyceridemia (HTG) is an independent risk factor for cardiovascular disease (CVD). Hemostatic variables [factor VII antigen (FVIIag), factor VII coagulant activity (FVIIc), activated factor VII (FVIIa), free and endothelial-associated (EC) tissue factor pathway inhibitor (TFPI) antigen, pre- and post-heparin total TFPI activity, EC-TFPI activity, prothrombin fragment 1 + 2 (F1 + 2), fibrinogen and D-dimer] were compared between 18 HTG patients and 20 controls to investigate whether HTG is associated with alterations in the extrinsic pathway and whether such alterations create a procoagulant state, as expressed by F1 + 2 and D-dimer levels. In addition, the effects of bezafibrate therapy (6 weeks, 400 mg/day) on these variables were studied in 18 HTG patients in a double-blind, placebo-controlled, cross-over study.

Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis.

Thrombin activatable fibrinolysis inhibitor (TAFI, or procarboxypeptidase B) is the precursor of a recently described carboxypeptidase that potently attenuates fibrinolysis. Therefore, we hypothesized that elevated plasma TAFI levels induce a hypofibrinolytic state associated with an increased risk for venous thrombosis. To evaluate this hypothesis, we developed an electroimmunoassay for TAFI antigen and used this assay to measure TAFI levels in the Leiden Thrombophilia Study, a case-control study of venous thrombosis in 474 patients with a first deep vein thrombosis and 474 age- and sex-matched control subjects.

Toward gene therapy for hemophilia A: long-term persistence of factor VIII-secreting fibroblasts after transplantation into immunodeficient mice.

Hemophilia A is caused by the lack of functional blood-clotting factor VIII. We have used retrovirus-mediated gene transfer to generate various cell lines, rodent as well as human, that secrete the human factor VIII protein. To study whether transplantation of genetically modified fibroblasts is a feasible approach for gene therapy of hemophilia A, we implanted the factor VIII-secreting cells into immune-deficient mice.

Activated protein C accelerates clot lysis by virtue of its anticoagulant activity.

The effect of human activated protein C (APC) on t-PA dependent fibrinolysis was studied in vitro using plasma (and whole blood) clot lysis techniques. Clot lysis was monitored by measuring the release of soluble 125I-labelled fibrin degradation products from the clot over time. It was demonstrated that the stimulatory effect of APC on plasma and blood clot lysis was specific for APC and depended on the presence of its active site and Ca2+ ions.

Expression of functional factor VIII in primary human skin fibroblasts after retrovirus-mediated gene transfer.

We have developed a retroviral-vector system for the transfer and expression of a cloned blood clotting factor VIII cDNA. Since inclusion of the complete cDNA into existing vectors is precluded by its large size, we deleted most codons for the B-domain, which is also excised during in vivo maturation of factor VIII. When inserted into the retroviral vector M5-neoR (Laker, C.

A second plasma inhibitor of activated protein C: alpha 1-antitrypsin.

Inactivation of activated protein C (APC) in normal human plasma was studied in the absence and presence of heparin. In the absence of heparin APC inactivation followed pseudo-first order kinetics. In the presence of heparin the neutralization of APC was found to be biphasic.

Immunoradiometric assays for human coagulation factor VII using polyclonal antibodies against the Ca(II)-dependent and Ca(II)-independent conformation.

Human coagulation factor VII is a trace plasma protein belonging to the vitamin K-dependent factors. Two specific and sensitive immunoradiometric assays for factor VII were developed using immunopurified rabbit antibodies against the Ca(II)-independent and Ca(II)-dependent conformation of factor VII. Both assays were insensitive to the activation state of factor VII.

Evaluation of the cuticle bleeding time in canine haemophilia A.

In this paper we describe our clinical experience and results with the cuticle bleeding time test in a colony of cross-bred Labrador retrievers with severe haemophilia A. The dogs have a severe bleeding tendency with a high incidence of fatal haemorrhages in the central nervous system. Homozygous females appeared to be especially prone to this lethal complication.

Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium.

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC.

Coagulation factors in the premature infant born after about 32 weeks of gestation.

In plasma samples from 10 premature infants born after about 32 weeks of gestation, a number of coagulation factors have been determined. For 9 infants, who were healthy, mean values are given: fibrinogen-antigen, 311 mg/dl; factor II, +/- 0.46 U/ml; factor V, 0.

Heterogeneity of haemophilia A: a study with three different antisera.

One human and one rabbit antibody against VIII:C--in a fluid-phase, inhibitor neutralization assay (INA)--and one human antibody--in a solid-phase, immunoradiometric assay (IRMA)--were used to investigate a group of 59 patients with severe, moderate and mild haemophilia A. Patients were classified as haemophilia A+ when the VIII:C/VIIIAG ratio was less than 0.4 while the absolute VIII:C antigen (VIIICAG) value exceeded 0.

Ontogenetic aspects of immune-complex trapping in the spleen and popliteal lymph nodes of the rat.

An ontogenetic approach was used to obtain information about the relation between structure and function of lymphoid tissues. In particular the development of the capacity to trap immune complexes was studied in relation to the development of the lymphoid compartments. For this purpose isologous horseradish (HRP)-ant-HRP complexes were injected into neonatal rats, and their fate was studied in the spleen and popliteal lymph nodes.

A monoclonal antibody to VIII:C produced by a mouse hybridoma.

Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody.

Immunoradiometric assay of procoagulant factor VIII antigen (VIIICAG).

An immunoradiometric assay of procoagulant factor VIII antigen was developed using a human antibody to the procoagulant activity of factor VIII (VIII:C). The assay measures specifically the antigen related to factor VIII procoagulant activity (VIIICAG) both in plasma and in serum. VIII:C and VIIICAG were measured in a group of healthy individuals and in a group of haemophilia A patients.