Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

Antithrombotic therapy for venous thromboembolic disease.

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in a minority of cases. Based on the hypothesis that a poor anticoagulant response to activated protein C (APC) would predispose to thrombosis, a set of new coagulation assays was developed that measure the anticoagulant response in plasma to APC. A middle-aged man with a history of multiple thrombotic events was identified. The addition of APC to his plasma did not result in a normal anticoagulant response as measured by prolongation of clotting time in an activated partial thromboplastin time (APTT) assay. Four of the proband's relatives had medical histories of multiple thrombotic events, and they and several other family members responded poorly to APC in the APTT-based assay. Subnormal anticoagulant responses to APC were also found in factor IXa- and Xa-based assays. Several possible mechanisms for the observed phenomenon were ruled out, such as functional protein S deficiency, a protein C-inhibitory antibody, or a fast-acting protease inhibitor against APC. Moreover, restriction fragment-length polymorphism analysis excluded possible linkage of the underlying molecular defect to factor VIII and von Willebrand factor genes. We now describe a previously unrecognized mechanism for familial thromboembolic disease that is characterized by poor anticoagulant response to APC. This would appear to be explained best by a hypothesized inherited deficiency of a previously unrecognized cofactor to APC. As we have identified two additional, unrelated cases with thrombosis and inherited poor anticoagulant response to APC, this may constitute an important cause for familial thrombophilia.

https://www.pnas.org/content/pnas/90/3/1004.full.pdf

Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.

Hemophilia is a rare disorder that is complex to diagnose and to manage. These evidence-based guidelines offer practical recommendations on the diagnosis and general management of hemophilia, as well as the management of complications including musculoskeletal issues, inhibitors, and transfusion-transmitted infections. By compiling these guidelines, the World Federation of Hemophilia aims to assist healthcare providers seeking to initiate and/or maintain hemophilia care programs, encourage practice harmonization around the world and, where recommendations lack adequate evidence, stimulate appropriate studies.

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Guidelines for the management of hemophilia.

In Sweden, prophylactic treatment of boys with severe haemophilia has been practised since 1958 in an attempt to convert the disease from a severe to a milder form. The present study population consisted of 60 severe haemophiliacs (52 A, 8 B), aged 3-32 years. Treatment is started when the boys are 1-2 years of age, the regimens used being 24-40 IU F VIII kg-1 three times weekly in haemophilia-A cases (i.e. greater than 2000 IU kg-1 annually) and 25-40 IU F IX kg-1 twice weekly in haemophilia-B cases. The orthopaedic and radiological joint scores (maximum scores of 90 and 78, respectively) are evaluated as recommended by the World Federation of Haemophilia. Of those subjects aged 3-17 years, 29 out of 35 individuals had joint scores of zero. The oldest group had only minor joint defects. The VIII:C and IX:C concentrations had usually not fallen below 1% of normal. All 60 patients are able to lead normal lives. In conclusion, it appears to be possible to prevent haemophilic arthropathy by giving effective continuous prophylaxis from an early age, and preventing the VIII:C or IX:C concentration from falling below 1% of normal.

Twenty-five years' experience of prophylactic treatment in severe haemophilia A and B

A new, exclusively radiologic classification of hemophilic arthropathy is based on a review of 54 patients who had had repeated radiologic examinations of all great joints before the institution of any specific treatment. In terms of a score, the severity of the arthropathy may be assessed in a single joint, in a single patient, or in a group of patients. Using the score system, the natural course of hemophilic arthropathy demonstrated that the severity of the arthropathy increased with age and degree of coagulation defect. All patients over 5 years of age with severe hemophilia were affected and the radiologic changes were progressive. There was a good correlation between the radiologically assessed severity of the arthropathy and the clinical findings.

A radiologic classification of hemophilic arthropathy.

Objectives. To review long-term prophylactic factor treatment in young patients with severe haemophilia A and B, focusing on the orthopaedic and radiological outcome.

Design. We received 34 patients with severe haemophilia A (n=29) and B (n=5), aged 7–22 years. Age at start of treatment was 1–4.5 years. Dosages of factor concentrate (F VIII and F IX, respectively) were 25–40 IU/kg body weight, three times a week for haemophilia A and twice a week for haemophilia B. The patients had been checked annually over a 5-year period (1990–95). Orthopaedic and radiological joint scores were evaluated according to recommendations by the World Federation of Haemophilia.

Setting. All results were obtained at the Department for Coagulation Disorders, University of Lund, Malmo University Hospital, Malmo, Sweden.

Results. Orthopaedic and radiological joint scores were found to have remained unchanged during follow-up in almost all patients and to be still zero (i.e. no unaffected joints) in 79% (n=27) of the patients.

Conclusion. There is a growing international consensus haemophilic arthropathy can be prevented by administering early high-dose prophylaxis. The results of the present investigation strongly support this opinion.

Haemophilia prophylaxis in young patients–a long‐term follow‐up

Fibrinolytic components after venous occlusion and concentrations of tissue plasminogen activator inhibitor were studied in 100 consecutive patients with confirmed recurrent deep vein thrombosis or pulmonary embolism. After 20 minutes of venous occlusion the fibrinolytic response was decreased in 33 patients, as measured both amidolytically with S-2251 and on fibrin plates. Two different mechanisms responsible for the poor fibrinolytic response could be distinguished. Twenty two of the patients in whom the response was poor released normal amounts of tissue plasminogen activator antigen, as assayed by immunoradiometric assay, but had appreciably increased concentrations of tissue plasminogen activator inhibitor. The 11 other patients in whom the response was poor had both low tissue plasminogen activator activities and low tissue plasminogen activator antigen concentrations but normal concentrations of tissue plasminogen activator inhibitor. The results show not only that defective synthesis or release of tissue plasminogen activator may be important in the pathogenesis of venous thrombosis but also that a large group of patients with thrombosis have an increased concentration of the inhibitor to tissue plasminogen activator.

https://www.bmj.com/content/bmj/290/6480/1453.full.pdf

Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor

Inga Marie Nilsson

Papers

Twenty-five years' experience of prophylactic treatment in severe haemophilia A and B

A radiologic classification of hemophilic arthropathy.

Haemophilia prophylaxis in young patients–a long‐term follow‐up

Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor

On an Inherited Autosomal Hemorrhagic Diathesis with Antihemophilic Globulin (AHG) Deficiency and Prolonged Bleeding Times1