A collaborative study to establish the 1st International Standard for factor XIII plasma.

BACKGROUND: An international collaborative study, involving 23 laboratories, was carried out, under the auspices of the FXIII Standardization Working Party (SWP), to calibrate the 1st International Standard (IS) for factor XIII (FXIII) plasma. METHODS: Potency estimates for the proposed candidate FXIII plasma (preparation Y: NIBSC code 02/206) were calculated relative to locally collected normal plasma pools (pool N), for both FXIII activity and antigen levels. RESULTS: Estimates of FXIII activity potency for preparation Y showed good agreement between laboratories, with an interlaboratory geometric coefficient of variation (GCV) of 11.5% and a mean value of 0.91 U mL(-1). Furthermore, there was a negligible difference in potencies by two commercially available methods, the potencies differing only by approximately 1%. Estimates of FXIII antigen (A(2)B(2) complex) potency for preparation Y showed good agreement between laboratories, with an interlaboratory GCV of 16.3% and a mean value of 0.93 U mL(-1). Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity (and antigen) per year of< 0.06% at the recommended storage temperature of -20 degrees C. CONCLUSIONS: The suitability and potency of preparation Y were considered by the participants, members of the ISTH/SSC FXIII Subcommittee, the Scientific and Standardization Committee and the SWP. Following their approval, preparation Y was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 1st IS for FXIII plasma with an activity potency of 0.91 IU per ampoule and an antigen potency of 0.93 IU per ampoule.

Characterization of cell-associated plasminogen activation catalyzed by urokinase-type plasminogen activator, but independent of urokinase receptor (uPAR, CD87).

The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 10(6) to 10(7) cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.

Regulation of tissue plasminogen activator activity by cells. Domains responsible for binding and mechanism of stimulation.

A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.

The effect of interleukin-1 on venous endothelium--an ultrastructural study.

The effect of systemic interleukin-1 (IL-1) on venous endothelium in the presence and absence of stasis has been studied by scanning electron microscopy (SEM). Recombinant human IL-1 beta at a concentration of 1 micrograms/kg or saline was injected intravenously into rabbits and allowed to circulate for 0.5 or 4.0 h after which complete stasis was induced for 1 h in an isolated segment of each jugular vein. One vein segment was then excised and the contents examined macroscopically for thrombi, while the other segment was fixed for SEM examination. When examined by SEM the endothelium from rabbits injected with IL-1 beta was perturbed with increased surface microvilli, blebs and gaps at cell junctions when compared with saline controls. Fibrin deposition was also observed after IL-1 beta, as was the adherence of essentially non-activated platelets to intact endothelium. However, macroscopic thrombi were not formed in isolated vein segments. We conclude that although fibrin strands and platelets were deposited on the endothelium, IL-1 is not a sufficiently powerful procoagulant stimulus to lead to an occlusive thrombus in acute experiments.

Potentiation of the antithrombotic action of dermatan sulphate by small amounts of heparin.

We have examined the effect in impairing thrombus formation of a preparation of dermatan sulphate (DS) alone and DS plus small amounts of unfractionated heparin (UFH). In rabbits given a dose of 150 micrograms/kg of DS alone, there was minimal reduction in serum-induced stasis thrombosis (Wessler model), with an overall score of 92.5% (100% = no impairment of thrombus formation). When the same dose of DS containing UFH was given (two different subgroups given DS containing 0.25 and 2.5% heparin by dry weight, respectively), the overall thrombus score was reduced to 60% (P less than 0.003), with no significant difference between the two subgroups. To achieve a comparable result with DS alone, a dose of 1 mg/kg was required. We conclude that very small amounts of UFH significantly enhance the antithrombotic action of DS, by a mechanism that has yet to be determined.

The relative antithrombotic effectiveness of heparin, a low molecular weight heparin, and a pentasaccharide fragment in an animal model.

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes' stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes' stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.

Relative efficacy of heparin and related glycosaminoglycans as antithrombotic drugs.

In a standardized animal model, unfractionated heparin (UFH) prevents venous thrombogenesis at a dose of 80 micrograms/kg. Oligosaccharide fragments of heparin, with very high anti-Xa activity both in vitro and in ex vivo plasma samples were less effective than UFH in preventing thrombosis. A decasaccharide fragment was virtually inactive in impairing thrombosis at this dose, although a 20-22 monosaccharide fragment showed some impairment. Dermatan sulfate, which has no anti-factor Xa activity, partially impairs both thrombin generation and stasis thrombosis. However, dermatan sulfate could not suppress thrombin generation below about 35% of control at the doses studied. Neither oligosaccharides nor dermatan sulfate were as effective on a weight basis as UFH in impairing thrombosis, particularly after 20 minutes' stasus. Maximal antithrombotic effects are achieved when both factor Xa and thrombin are inhibited. Drugs which act primarily on factor Xa (oligosaccharides) or thrombin by non-ATIII pathways (dermatan sulfate) are less efficient than UFH as antithrombotic drugs.

Heparin and bleeding: an association with lipase release.

The effects of four sulphated polysaccharides on bleeding time and lipase release in rabbits have been compared. Unfractionated heparin (UFH) and pentosan polysulphate both gave significant prolongation of bleeding times and high lipase release. Low molecular weight heparin had reduced effects on bleeding time and lipase release, while dermatan sulphate had no influence on either parameter. There was a highly significant correlation (r = 0.97) between these two measurements. These results suggest that the same structural features influence both the haemorrhagic and lipase-releasing properties of sulphated polysaccharides.

Experimental studies on the relative efficacy of dermatan sulphate and heparin as antithrombotic agents.

In this study, the anticoagulant and antithrombotic properties of unfractionated heparin (UFH) and dermatan sulphate (DS) were compared. The ability of UFH and DS to impair thrombin generation in vitro and in ex vivo plasma samples was also studied. DS has minimal anticoagulant activity by conventional assays but impairs thrombin generation both in vitro and in ex vivo plasma samples. However, thrombin generation could not be suppressed below about 35% of control values at all doses of DS studied. While this was sufficient to impair experimental venous thrombosis during 10 minutes' stasis, DS was ineffective in preventing thrombosis following 20 minutes' stasis in doses up to 1.25 mg/kg. In contrast, 1 microgram/ml of UFH completely suppressed thrombin generation in vitro, and 150 micrograms/kg prevented thrombogenesis over a period of 20 minutes' stasis. Neither drug prolonged the bleeding time (BT) at effective antithrombotic doses, but 2.5 mg/kg UFH significantly increased the BT, whereas DS did not. While DS has antithrombotic activity, it is less effective than UFH in inhibiting thrombin generation, and as an antithrombotic agent.

Anticoagulant activities of pentosan polysulphate (Hémoclar) due to release of hepatic triglyceride lipase (HTGL).

Subcutaneous injections of 50 mg pentosan polysulphate (Hémoclar) were given to normal volunteers and the effects on anti-Factor Xa activity, thrombin generation and lipase release measured. Concentrations of pentosan polysulphate were measured by a competitive binding assay and the mean peak level found to be 1.6 micrograms/ml. Anti-Xa clotting activity rose to 0.034 iu/ml and thrombin generation induced by lipid peroxides was inhibited by approximately 50%. Neither of these effects could be accounted for by the direct action of pentosan polysulphate at the concentrations measured. Pentosan polysulphate was very effective in releasing lipase, approximately 70-80% of the total enzyme activity being due to hepatic triglyceride lipase (HTGL). In vitro addition of purified HTGL to plasma markedly enhanced anti-Xa clotting activity, and caused a 70% inhibition of lipid peroxide induced thrombin generation. Anti-Xa activity of post-injection plasma was increased rather than neutralised by addition of polybrene, and this effect could be mimicked by addition of polybrene to plasma containing pentosan polysulphate and purified HTGL. It is concluded that, when given in low doses subcutaneously, pentosan polysulphate acts as an indirect anticoagulant, its major effects being due to release of HTGL.

The relationship between vessel wall injury and venous thrombosis: an experimental study.

The relative importance of stasis, vessel wall damage and hypercoagulability in the pathogenesis of venous thrombosis remains disputed. While the combination of local vascular stasis and systemic hypercoagulability can be shown to produce experimental thrombi within a few minutes, it has been claimed that vessel wall damage is also a necessary component of venous thrombogenesis. In this experimental study, mechanical crushing of the jugular veins produced patchy areas of denuded endothelium, with underlying vessel wall oedema, as seen by ultrastructural examination. While the exposed subendothelium became covered with activated platelets following restored blood flow, there was no fibrin formation after 5 min. When blood flow was restored for 60 min following the crush injury, white cells could be seen adhering to and migrating through the vessel wall, although there was still no visible fibrin. The addition of venous stasis for 20 min did not lead to the formation of stasis thrombi in association with the damaged areas. The present experiments demonstrate that, far from there being subtle endothelial damage contributing to acute venous thrombosis, even readily demonstrable damage is a poor stimulus to fibrin formation at local sites of vessel wall injury.

A comparison of heparin potency estimates obtained by activated partial thromboplastin time and british pharmacopoeial assays.

Heparin samples from five manufacturers were assayed by the revised British Pharmacopoeia (BP) heparin assay and the results compared with those obtained using the activated partial thromboplastin time (APTT) assay. The United States Pharmacopoeia (USP) reference heparin preparation and the 4th International Standard (IS) for heparin were also assayed by the two methods relative to the 3rd IS. The results obtained by the revised BP assay were in close agreement with those obtained by the APTT assay for all the heparins that were tested. The assays revealed that there is at least a 10% discrepancy between the International Unit for heparin and the USP unit.

Low-affinity heparin potentiates the action of high-affinity heparin oligosaccharides.

Previous studies have shown that high-affinity (HA) heparin oligosaccharides, with molecular weights of 3,000-5,000, were less effective than unfractionated heparin in preventing serum-induced venous thrombosis in rabbits, using a Wessler stasis model. In the present study, a larger high-affinity fragment (M.Wt. 6,000-6,500) was also found to be less effective than unfractionated heparin as an antithrombotic agent. However, addition of 80 micrograms/kg low affinity (LA) heparin to 80 micrograms/kg of this HA fragment significantly potentiated its antithrombotic activity, and the antithrombotic action of the mixture was equivalent to that of unfractionated heparin. Significant potentiation of antithrombotic activity was also observed on the addition of LA heparin to a HA decasaccharide (M.Wt. 3,000-3,500) with anticoagulant activity only against Factor Xa. The LA heparin content of low molecular weight heparin fractions appears to be an important determinant of their antithrombotic activity.

High and low affinity heparin compared with unfractionated heparin as antithrombotic drugs.

A preparation of heparin was separated by affinity chromatography into two fractions: one of high ( HAH ) and the other of low (LAH) affinity to antithrombin III. These two fractions were compared with unfractionated heparin ( UFH ) by in vitro assay and their ability to impair experimental stasis thrombosis was also examined. Although the in vitro activity of HAH was double that of UFH , HAH was less effective than UFH as an antithrombotic drug; LAH was virtually inactive, both in vitro and in vivo. A mixture of 30 micrograms/kg of HAH and 50 micrograms/kg of LAH was as effective in preventing thrombosis as 80 micrograms/kg of UFH , and was more effective than 40 micrograms/kg of HAH alone, demonstrating that LAH potentiates the action of HAH in vivo.

The effect of stasis on the venous endothelium: an ultrastructural study.

In carefully dissected neck veins, no evidence was found of platelet adherence to the vessel wall or leucocyte migration. However, 30-60 min of total stasis led to polymorphonuclear leucocytes sticking to the endothelium and their subsequent migration. This migration across the vessel wall resulted from stasis and not the trauma of dissection. Adherence and migration of leucocytes did not cause gross endothelial cell damage or desquamation within the observed period of stasis and there was no associated platelet adherence following restoration of blood flow. Thus leucocyte migration does not impair the non-thrombogenicity of the endothelium in acute experiments.

A low molecular weight heparin compared with unfractionated heparin.

A 6000 daltons low molecular weight heparin (LMWH) was compared with unfractionated mucosal heparin in vitro and in vivo. Despite unimpressive specifications by clotting assays in vitro, the LMWH gave high and sustained activity in vivo by anti-Factor Xa assays, following subcutaneous injection. However, activity measured by APTT and calcium thrombin time assays was at least as high as occurred following unfractionated heparin. On the basis of clotting assays, there seems no reason to expect a lower incidence of haemorrhagic side-effects following the clinical use of this LMWH. The study also strikingly demonstrates the inadequacy of in vitro clotting assays for assessing the in vivo behaviour of LMWH.

Effects of heparin oligosaccharides with high affinity for antithrombin III in experimental venous thrombosis.

The in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 microgram/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays. It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Resistance of normal endothelium to damage by thrombin.

We examined the effect of locally infused thrombin on rabbit neck veins, using autologous [111In]indium-labeled platelets on the vessel wall. However, when the animals were pre-treated with aspirin (10 mg/kg), there was a marked change in the ratio of radioactive counts between control and treated segments, consistent with platelet deposition on the walls of thrombin-treated segments. Thrombin-treated and control veins were also examined by transmission and scanning electronmicroscopy. Although occasional clumps of platelets were seen adhering to the vessel wall in both control and treated segments, the endothelium was essentially intact. There was no evidence of substantial denudation of the endothelium, and overall there was little morphological difference between control and treated segments. It is concluded that normal endothelium is not damaged by thrombin, and that venous thrombi develop by a direct effect of thrombin on platelets and fibrinogen, and not by thrombin-mediated damage to the vein wall.

A comparison of pentosan polysulphate and heparin. II: Effects of subcutaneous injection.

A comparison has been made between the effects of pentosan polysulphate (SP54) and mucosal heparin following subcutaneous injection in man. Unlike heparin, pentosan polysulphate has relatively little effect in vivo as measured by anti-factor Xa clotting assay and none by an anti-Xa amidolytic assay (S-2222). However, pentosan polysulphate is at least as potent as heparin on a weight basis in producing activation of lipoprotein lipase, shortening of the euglobulin clot lysis time and impairing the generation of factor Xa. Our data indicate that pentosan polysulphate has more marked effects in vivo than in vitro, that the action of the drug on clotting is mediated mainly via an At III-independent pathway, and that its effects are not confined to the coagulation system.