In vivo penetration of experimentally produced clots by monoclonal antibodies.

Antifibrin monoclonal antibodies show potential as clot targeting agents for diagnosis and possibly therapy in thrombotic disease. To be effective the antibody must bind to the fibrin component of the clot. The ability of two antifibrin mabs (NIB 1H10 and NIB 12B3) to penetrate occlusive clots in vivo was investigated. Both mabs react with human fibrin but not with human fibrinogen nor with the fibrin or fibrinogen from the species used in this study. Two heterologous animal (sheep and rabbit) thrombus models were used. Clots in both cases were made within isolated vein segments using a mixture of human and native fibrinogen. The clots in sheep veins were observed radiographically and found to be occlusive for a mean of 4.2 +/- 2.2 days and thereafter appeared only partially occlusive. When targeted in their occlusive phase (131)I labelled mab accumulated in the clot reaching a maximum ratio of 1.82 +/- 0.42 when compared to counts in homologous sheep clots in the contralateral limb. It was confirmed in the rabbit jugular vein model that total occlusivity did not prevent antibody accumulation in the heterologous clot by injecting the fibrin specific mab 1H10 and examining the clot excised after 1, 6 and 24 h using immunofluorescence. In a further series of similar experiments (125)I labelled mab 1H10 was used and detected using autoradiography. Both sets of experiments indicated that penetration of occlusive clots by the antibody occurred and that considerable accumulation was present at 6 and 24 h. The results indicate that a circulating antibody can readily gain access to experimentally produced clots in occluded veins.

The haemostatic balance -- Astrup revisited.

Prof. Tage Astrup first elaborated the notion that blood fluidity involved a balance between the tendency of blood to clot and for such clots to lyse. It would seem that, at that time, this haemostatic balance involved the notion that forming fibrin orchestrated its own destruction by stimulating fibrinolytic activity. In this review, we have clarified the detail of this balance and developed the thesis that Astrup's far-sighted balance notions involve a variety of control mechanisms. These involve the notion that thrombin, being at first sight a procoagulant, can also, in conjunction with thrombomodulin, act as a stimulus of anticoagulant activity by the generation of activated protein C. The thrombin-activatable fibrinolytic inhibitor (TAFI) is also involved in this balance since the generation of thrombin provokes the neutralisation of fibrinolysis by the TAFI pathway. The kallikrein/factor XII/urokinase pathway is discussed indicating yet another aspect of balance between the generation of coagulation and fibrinolysis. The overall theme of this review, apart from an insight into various aspects of the haemostatic balance, is that blood has a strong tendency to clot when tissue is damaged, and the intact vasculature requires major anticoagulant systems to prevent clots adhering to and stabilising in the vasculature.

A pig collagen peptide fraction. A unique material for maintaining biological activity during lyophilization and during storage in the liquid state.

There is frequent use of human and animal proteins as stabilizers during lyophilization of a variety of biological substances with a view to long term stable storage. This report describes the comparative excellent stabilizing effect of a porcine collagen peptide fraction (CPF) during the lyophilization and subsequent storage of three commonly used biological substances, alkaline phosphatase, tissue plasminogen activator and thrombin. The CPF was heated to 150 degrees C for one hour before use. The CPF was shown to have some advantage during lyophilized storage over human serum albumin. Solutions of thrombin stored in CPF at room temperature and at 37 degrees C for one week retained nearly all activity, while storage of thrombin in human serum albumin solution at 37 degrees C lost nearly all biological activity. These preliminary data suggest that porcine CPF is a safe and advantageous stabilizer for addition to biological products with a view to long-term lyophilized storage and short-term liquid storage.

The international standard for plasminogen activator inhibitor-1 (PAI-1) activity.

Since the finding that plasminogen activator inhibitor-1 (PAI-1) may influence the initiation and progression of acute myocardial infarction, the assay of PAI-1 in plasma using a variety of commercial kits has become commonplace. The need for a standard to define the activity of PAI-1 prompted an international collaborative study (ICS) to calibrate the functional potency of a lyophilised plasma PAI-1 preparation (92/654). Since PAI-1 inhibits the 2 major plasminogen activators, tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) in an equimolar manner it was important to establish the potency of the PAI-1 inhibitor in terms of both t-PA and u-PA neutralisation. While the ICS indicated a wide spread of data between the laboratories the mean value of 27.5 t-PA neutralisation units and 7.0 u-PA neutralisation units was confirmed by numerous assays at NIBSC using a tedious but technically reliable titration assay procedure. The plasma PAI-1 proposed standard (92/654) was stable at 20 degrees C for 20 months. The Fibrinolysis Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH), (meeting in Leuven. Belgium in September 1994) has recommended that the plasma PAI-1 (92/654) should be accepted as the International Standard for PAI-1 and should define a unitage in terms of both t-PA and u-PA neutralisation. Subsequently the Expert Committee on Biological Standardization of the World Health Organization (ECBS-WHO) meeting in Geneva, Switzerland in October 1995 approved plasma PAI-1 (92/654) as the International Standard.

Detection of fibrin in canine neoplasia.

A murine monoclonal antibody, designated 1H10, produced using a human fibrin-related immunogen, was shown to bind avidly to dog fibrin, but not to dog fibrinogen. Using immunofluorescence, fibrin was detected in canine gastric adenocarcinoma and in mixed tissue from a mammary tumour. No fibrin could be detected in bronchogenic carcinoma tissue.

The International and "NIH" units for thrombin--how do they compare?

The current International Standard (IS) for alpha-thrombin was established in 1991. It was related in unitage with the 1st IS for Thrombin established in 1975 and contains 100 IU of thrombin activity. The National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents and this study reports on a comparison of the thrombin units defined by these 2 standards. This study has indicated that one "NIH" unit is equivalent to 1.1 to 1.3 IU of thrombin, depending on the influence of PEG in the assay. A unitage ratio figure of 1.15 was recommended following analysis of data obtained in the presence and absence of PEG in the comparative assays. This was confirmed by amidolytic assay data.