Alan N. Whitaker

An immunoassay for human D dimer using monoclonal antibodies

Monoclonal antibodies (MAb) were raised against human D dimer. The hybridomas were screened with a solid phase enzyme immunoassay against D dimer and fibrinogen degradation products. Among the panel of MAb identified, two distinct patterns emerged; the majority belonging to a panspecific class reacting against epitopes present on both D dimer and fibrinogen degradation product Dcate and a monospecific class reacting with determinants apparently present only on D dimer. A number of MAb were further characterised for their ability to specifically capture antigen in a solid phase enzyme immunoassay and assays were developed which have a sensitivity of 10 ng/ml for D dimer or crosslinked fibrin derivatives and may be suitable for detection of crosslinked derivatives in serum and plasma samples in a clinical situation.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom ofPseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant,Ki ≈ 10−11 mol/l), while both of the latter bound less tightly (Ki ≈ 10−9 mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan,Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Summary. Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as antihaemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full‐length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz‐type serine protease inhibitors. When expressed as glutathione S‐transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with Ki values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Identification of D dimer-E complex in disseminated intravascular coagulation

Abstract Serum fibrin degradation products in a patient with severe disseminated intravascular coagulation (caused by fulminant pneumococcal sepsis), were characterized using immunoprecipitation, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and crossed immunoelectrophoresis. These revealed a spectrum of fragments identified as high molecular weight (HMW) complexes, a component with mobility on SDS PAGE similar to that of fibrinogen X (“X”), D dimer and E. By their electrophoretic characteristics and reactions with antisera to fragments E and D it was found that most of the D dimer and E were noncovalently complexed as D dimer-E, and that there was relatively little free D dimer and free E. This pattern of FDP (HMW complexes, “X” and D dimer-E) has also been identified during the lysis of crosslinked fibrin by plasmin. The HMW complexes and “X” are believed to be crosslinked X oligomers and crosslinked Y-Y or Y-D respectively.

Plasma cross linked fibrin degradation products in pulmonary embolism.

Plasma concentrations of cross linked fibrin degradation products, a marker of intravascular thrombosis and fibrinolysis, were measured in 495 patients with suspected pulmonary embolism referred for ventilation-perfusion lung scanning to determine whether concentrations are increased in pulmonary embolism and their potential use in diagnosis. Lung scans were described as normal (n = 66) or as showing a low (n = 292), indeterminate (n = 58), or high probability (n = 79) of pulmonary embolism. There was a difference between the mean levels of cross linked fibrin degradation products in each scan category: normal scans, 142 ng/ml; low probability scans, 295 ng/ml; indeterminate probability scans, 510 ng/ml; high probability scans, 952 ng/ml (p less than 0.001). Of the patients with high probability scans, 96% had raised concentrations. Explanations for discrepant low results include incorrect scan diagnosis, delay in blood sampling, and anticoagulation. Of the patients with a low or indeterminate probability of pulmonary embolism, 43% had increased concentrations of cross linked fibrin degradation products that could be attributed in most cases to another illness. Owing to the wide range of values in each lung scan diagnostic category, raised concentrations of these fibrin degradation products cannot be used without reference to the patients clinical state as a discriminatory test for pulmonary embolism. Further evaluation of the significance of normal concentrations in excluding a diagnosis of pulmonary embolism appears to be warranted.

A Novel Serine Protease Inhibitor from the Australian Brown Snake, Pseudonaja textilis textilis: Inhibition Kinetics

A 7kDa protein component, isolated from the venom of the Australian brown snake, Pseudonaja textilis textilis, was found to be an inhibitor of the serine protease plasmin. Its mode of action, inhibitory efficiency and specificity were determined, and compared with those displayed by aprotinin (a Kunitz inhibitor). Using aprotinin as a model small protein protease inhibitor, and with plasmin as the enzyme, the observed slow-onset inhibition was consistent with the two-stage reversible mechanism, E+IEIEI′. Formation of the initial complex (EI) was fast, but binding was relatively loose with an initial K=3.78 nM, while transition from EI to EI′, was slow, and binding was tight, with a final K′=53.2 pM. With snake inhibitor, and in contrast to the above standard mechanism, the observed inhibition was consistent with a competitive, single-stage reversible mechanism, prior to cleavage of the inhibitor to an inactive product. Plasmin and trypsin bound the snake inhibitor via this mechanism, with K values of 0.15 μM and 0.30 μM, respectively. Snake protein concentrations up to 1.0 μM failed to inhibit a number of serine proteases, including recombinant two-chain tissue plasminogen activator, high molecular weight urokinase (55 kDa), α thrombin, elastase and α chymotrypsin. Results demonstrate that the small protein protease inhibitor from the Australian brown snake does not act via the standard slow, tight-binding mechanism common to other small protein serine protease inhibitors.

The binding of glu- and lys-plasminogens to fibrin and their subsequent effects on fibrinolysis

The affinities of glu- and lys-plasminogens (plgn) for totally crosslinked fibrin were compared in two systems: a) incorporation into fibrin during clotting and b) direct adsorption onto fibrin immersed in plgn solution. Over a wide range of concentrations of plgn, uptakes were greater with incorporation than immersion, and in purified than in plasma systems. In all cases lys-plgn showed a greater affinity for fibrin than did glu-plgn. The potencies of glu- and lys-plgn were also compared in lysis systems using 125I-labelled fibrin. The lysis achieved increased in proportion to the plgn concentrations to which clots were exposed. Fibrin to which plgn was adsorbed by immersion lysed slowly and incompletely when transferred to streptokinase (SK), irrespective of the type of plgn used. However, more lysis was achieved after incorporation of plgn, in which system prior exposure to lys-plgn gave more lysis in SK than did glu-plgn. In contrast, a third and slightly more potent lysis system (adsorbing SK-activator to washed fibrin then transferring the fibrin to plgn solution, called the SK→plgn regimen), produced marginally more lysis in glu- than lys-plgn. The molecular species of fibrin degradation products obtained with different lysis regimens were not altered by interchanging glu- and lys-plgns. Again, with either type of plgn, the binding to fibrin (in both incorporation and immersion systems) and the lysis rates of fibrin (in the SK→plgn regimen), were both decreased in the presence of plasma or serum. The pertinence of these test-tube data to in vivo requirements for thrombolytic therapy is speculative, but they support both current theories that the impetus to fibrinolysis is provided a) by intrinsic plgn and b) by the adsorption of plgn activator to fibrin. It cannot be assumed that lys-plgn must be more effective than glu-plgn in lysis systems because of superior affinity for purified fibrin, and in these studies the most active lysis was achieved when either type of plgn percolated the fibrin and was activated to plasmin in situ.

Focused antithrombotic therapy: Novel anti-platelet salicylates with reduced ulcerogenic potential and higher first-pass detoxification than aspirin in rats☆

The use of aspirin as an anti-platelet drug is limited by its propensity to induce gastric injury and by its adverse effect on vascular prostacyclin formation. Two phenolic non-steroidal anti-inflammatory drugs (salicylic acid and diflunisal) were modified by esterification with a series of O-acyl moieties. The short-term ulcerogenic in vitro and in vivo anti-platelet properties, pharmacodynamic profiles, and extent of hepatic extraction of these phenolic esters were compared with aspirin (acetylsalicylic acid). The more lipophilic esters (longer carbon chain length in O-acyl group) show significantly less gastrotoxicity in stressed rats than does aspirin after a single oral dose. The in vitro and in vivo anti-platelet studies show that these phenolic esters inhibited (1) arachidonate-triggered human platelet aggregation and (2) thrombin-stimulated rat serum thromboxane A2 production by platelets in the clotting process almost as effectively as aspirin. The hepatic extractions of these O-acyl derivatives are significantly higher than those of aspirin. The pharmacodynamic studies show that these O-acyl derivatives of salicylic acid and diflunisal probably bind to, or combine with, the same site on the platelet cyclooxygenase as aspirin. Replacing the O-acetyl group with longer chain O-acyl moiety in this series of phenolic esters markedly reduced the potential of these agents to induce short-term gastric injury but did not lessen their activity as inhibitors of platelet aggregation. These non-acetyl salicylates may therefore represent a novel class of anti-platelet drugs with less ulcerogenic potential.

Fibrinolysis as a feature of disseminated intravascular coagulation (DIC) after envenomation

Blood was obtained from four patients envenomated by the Australian common brown snake, Pseudonaja textilis textilis. This elapid snake has one of the most toxic venoms in the world, containing extremely potent neurotoxic and coagulant components. The latter is a potent complete prothrombinase, converting prothrombin to alpha-thrombin, and comprises more than 30% of the total venom protein. The four envenomated patients developed a typical consumption coagulopathy. Serial serum and plasma samples from patients were studied by immunoaffinity adsorption, 2-alanine precipitation of fibrinogen and fibrinogen-related products and 2-dimensional immunoelectrophoresis, and assayed for crosslinked fibrin degradation products as D dimer, using the monoclonal antibody, DD-3B6/22. These procedures showed the virtually complete disappearance of fibrinogen, accompanied by the appearance of large quantities of fibrinogen and fibrin degradation products consisting of both crosslinked and noncrosslinked species. With recovery, a homogeneous high molecular weight fibrinogen was observed. The data suggest that the prothrombin activator of this venom causes the generation of thrombin which subsequently converts fibrinogen to fibrin and stimulates partial crosslinking of both alpha and gamma-chains. The resultant disseminated intravascular coagulation is accompanied by very active secondary fibrinolysis which apparently limits the extent of any microvascular thrombosis but which may contribute to a bleeding tendency.