Chromogenic substrates in haemostasis.

Advances in knowledge of the structure-function relationships of the proteins involved in haemostatic pathways, have made it possible to synthesize small peptides which mimic the active site of many of the serine proteases concerned. Attachment to the cleavage site of such synthetic peptides, of a detector molecule such as para-nitroaniline which, when released, produces a coloured compound has enabled many of the enzyme reactions of haemostasis, and most of the co-factors and naturally-occurring inhibitors, to be individually and sensitively quantitated. Chromogenic substrate assays are very specific and overcome the criticism levelled at many conventional assays that, being based on the speed of formation or the rate of destruction of a fibrin clot, they frequently involve enzymatic reactions in addition to that being examined. Moreover, chromogenic substrate assays are generally simple and quick to perform and are readily automated. They are thus economical of manpower and, more importantly from the point of view of patient care, they permit the rapidly-changing status of those with acute derangements of haemostasis to be monitored more frequently and comprehensively than is possible using some conventional assays. Chromogenic substrate assays have some limitations, however. Since they mimic only a small portion of the natural substrate, they may not be sensitive to structural defects elsewhere in the molecule, and may thus not totally reflect biological activity. Though technically simple to perform, the defined incubation times and temperatures must be rigidly adhered to if reliable results are to be obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of snake venom components on the haemostatic system.

Among the components in snake venom are a number which have profound effects (either stimulatory or inhibitory) on haemostatic mechanisms, including coagulation, fibrinolysis, platelet function and vascular integrity. As a consequence, human victims of snakebite may suffer severe and sometimes fatal haemorrhagic and/or thrombotic sequelae. Many of these venom components have been isolated and their precise mechanisms of action established. Apart from direct fibrinolysins, procoagulants predominate, most of these exerting their effect late in the clotting cascade, activating factor X or prothrombin or directly converting fibrinogen to fibrin. Some of the procoagulants are, or have the potential to be, used as therapeutic agents. Some venom components have been put to use as laboratory reagents for diagnostic purposes or for characterising molecular defects of haemostasis, although because they often have unphysiological actions, results must be interpreted with caution. These and other useful constituents e.g. protein C activator and platelet aggregating agents are discussed.

A study of the effect of ethamsylate (Dicynene) on the bleeding time, von Willebrand factor level and fibrinolysis in patients with von Willebrand's disease.

Nine patients with clinically moderate or severe Type I von Willebrand's disease were treated for 2 weeks with ethamsylate (2 g/day in four equal doses) and with a matched placebo in a randomised double-blind trial. Template bleeding time, von Willebrand factor activity (ristocetin co-factor) and antigen, euglobulin lysis time and type I tissue plasminogen activator inhibitor were determined before and at the end of each treatment period. None of these parameters showed any significant change attributable to ethamsylate. Thus, despite the fact that five patients thought subjectively that their bleeding symptoms improved during ethamsylate treatment compared to only one while on placebo, we obtained no evidence that the drug was of benefit to patients with von Willebrand's disease.

Platelet aggregation and plasma lipoproteins in alcoholics during alcohol withdrawal.

Platelet aggregation, platelet lipid composition and plasma lipoprotein concentrations were measured each week in a group of seventeen alcoholics, without overt liver disease, for one month, following acute, total alcohol withdrawal. The platelets were initially hypoaggregable but, within 1-2 weeks of cessation of drinking, they became hyperaggregable and then gradually returned towards normal values. Hyperaggregability could not be explained by increases in either the cholesterol or the arachidonic acid content of the platelets. Plasma very-low-density lipoprotein cholesterol levels remained high throughout the study, but the initially raised levels of high-density lipoprotein (HDL) cholesterol fell by 26%. Low-density lipoprotein (LDL) cholesterol concentration rose by 10% after two weeks of withdrawal but then returned to about the starting level. The resulting changes in the plasma LDL-cholesterol:HDL-cholesterol ratio, which had increased by more than 50% after two weeks of abstinence, essentially paralleled the time course of enhanced platelet reactivity in all but four of the alcoholics. These findings suggest that alterations in plasma lipoprotein concentrations during acute alcohol withdrawal may be a contributory factor to the haemostatic disorders present in such patients.

Studies on the action of ethamsylate (Dicynene) on haemostasis.

Twelve healthy subjects received ethamsylate or a placebo by mouth over a 48 h period in a randomized double-blind trial. The template bleeding time (including estimation of amount of blood loss), platelet aggregation studies, and plasma levels of plasminogen, alpha 2-antiplasmin and fibronectin were carried out before and during treatment. The effect of a single dose (600 mg) of aspirin, given 24 h after commencement of treatment, was also determined. Neither ethamsylate nor placebo caused a significant change in the basal values of any of the variables monitored but both the prolongation of the bleeding time and the amount of blood loss induced by aspirin were significantly less during ethamsylate treatment than with placebo. Ethamsylate failed to prevent the aspirin-induced elimination of the secondary wave of platelet aggregation. We conclude that ethamsylate may reduce the haemorrhagic symptoms associated with mild functional platelet defects.

Inhibitors of platelet aggregation in the fetoplacental unit and myometrium with particular reference to the ADP-degrading property of placenta.

Extracts of fresh tissue from the feto-placental unit and myometrium were tested for their ability to inhibit ADP-induced platelet aggregation and to degrade ADP. Placental extracts caused rapid reversal of aggregation and degraded ADP, both effects being mimicked by HPAP. However, whereas the latter was inhibited by L-phenylalanine but not by heating to 65 degrees C for 5 minutes, the reverse was true for crude placental extracts. Umbilical cord vessels and myometrium totally inhibited platelet aggregation in a similar way to pure PGI2. Both tissues also exhibited ADP-ase activity but were much less potent in this respect than placenta. In the system used, little or no anti-aggregatory activity was detected in extracts of non-vascular cord tissue, fetal membranes or amniotic fluid, although the two latter tissues had a weak ADP-degrading effect. Thus, it appears that in contrast to myometrium and umbilical cord vessels, the major inhibitor of platelet aggregation in placenta is an ADP-ase and not PGI2. While part of the inhibitory effect of placenta may be due to HPAP, other ADP-degrading enzymes also seem to contribute to the overall anti-aggregatory property of this organ.

Hemostatic evaluation in bleeding disorders from native blood. Clinical experience with the hemostatometer.

Primary hemostasis (PH), i.e., hemostatic platelet plug formation, and the subsequent coagulation were recorded and quantified from the same nonanticoagulated venous blood sample with the use of the Haemostatometer. In addition, platelet thrombus formation induced by interaction of flowing native blood with a collagen fiber under low shear rates (450 s-1) was simultaneously analyzed by this device. The effect of monoclonal antibodies (MoAbs) directed against von Willebrand's factor antigen (vWF:Ag), platelet glycoprotein Ib (GPIb) and the GPIIb/IIIa complex, and fibrinogen were studied. PH was significantly inhibited by MoAbs against vWF:Ag, GPIIb/IIIa, and fibrinogen but was unaffected by antibody against GPIb. Collagen-induced thrombosis was prevented by MoAbs against vWF:Ag and GPIb, slightly inhibited by antifibrinogen, and unaffected by blockage of platelet membrane GPIIb/IIIa. The effect of a single 600-mg dose of aspirin was monitored, and abnormal PH was still detectable five days later. From the 13 hemophiliacs tested, 7 showed significantly prolonged PH. In von Willebrand's disease, a characteristic defect of PH with significant inhibition or absence of collagen-platelet interaction was observed in all the 11 patients. PH was greatly prolonged in both of the two patients with storage pool deficiency. The technique detected improvement of platelet function, i.e., PH in all of six patients with bleeding disorders after replacement therapy or DDAVP infusion. The authors conclude that the Haemostatometer technique is a sensitive test for determining platelet dysfunction and monitoring efficacy of factor-replacement or DDAVP therapy.

Changes in the blood platelets of alcoholics during alcohol withdrawal.

The effects of alcohol withdrawal on platelet count and platelet function was studied sequentially in a group of alcoholics. Baseline values for platelet count, platelet adenine nucleotides and plasma beta-thromboglobulin (beta TG) level were within the normal range but platelet aggregability (especially with ADP and adrenaline) and circulating platelet aggregates were decreased for the group as a whole. After alcohol withdrawal there was a pronounced increase in all parameters measured which reached statistical significance in many cases and persisted for two to four weeks. The potential implications and possible mechanisms for these changes are discussed.

Haemostatic mechanism in uraemia.

The haemostatic mechanism was investigated in 20 patients with renal failure, of whom nine had evidence of a bleeding tendency. A defect of platelet function was the most common finding. The effect of dialysis on the bleeding state is briefly discussed, and a scheme for the routine investigation of haemostasis in renal failure is put forward.

Morphological and functional disturbances of platelets induced by cryopreservation.

In vitro morphological and functional studies were carried out on platelets which had been cryopreserved in the presence of 5% dimethyl sulphoxide (DMSO). Overall loss of platelets was around 50%. Those which survived freezing and reconstitution showed marked morphological deterioration, increase of procoagulant activity (PF3a) and a decrease in their aggregability and adenine nucleotide content. We conclude that if transfused, cryopreserved platelets are likely to be less effective than fresh platelets and may activate coagulation in vivo and that they should only be used when suitable fresh platelets are not available.

Platelet function in beta-thalassaemia major.

Abnormal platelet aggregation was found in eight (44%) of 18 patients with beta-thalassaemia major and transfusional iron overload. The aggregation defect bore no correlation with the degree of hepatic fibrosis, liver function tests, whether or not splenectomy had been performed, the degree of iron overload, haematocrit, platelet count, serum vitamin E level, or leucocyte ascorbate concentration. Only three of the 18 patients showed prolonged bleeding times as well as abnormal platelet aggregation, and only one of these suffered clinically significant haemorrhage. The results show that a proportion of patients with beta-thalassaemia major have abnormal platelet function. It is possible, however, that the in vitro abnormality might be due partly to artefacts induced by manipulations required to remove the abnormal thalassaemic red cells, and this may explain the much lower incidence of significant haemorrhage.

Platelet aggregation studies during transient hypoglycaemia: a potential method for evaluating platelet function.

The effect of acute hypoglycaemia on platelet function was examined in patients undergoing an insulin stress test. Enhanced platelet aggregation was observed in all cases but platelet count, platelet adenine nucleotides, and the plasma level of von Willebrand factor were unchanged overall. The onset of the hypoglycaemia-induced increase in platelet aggregation coincided with the lowest blood glucose levels recorded and with the clinical signs of adrenaline release. Increased platelet aggregation was maintained thereafter for the two-hour test period. There was no apparent correlation with changes in cortisol, growth hormone, and prolactin. No change in platelet function was observed after the administration of L-dopa. We suggest that the measurement of platelet aggregation during a standard insulin stress test may provide a means of evaluating platelet function in vivo and the influence of drugs thereon.

Assessment of platelet function in patients with Raynaud's syndrome.

Platelet function was studied in 11 patients with Raynaud's syndrome and 11 healthy controls. Platelets obtained from patients with Raynaud's syndrome were significantly more responsive to adrenaline, produced more thromboxane A2, and were resistant to prostaglandin inhibitors (prostacyclin and prostaglandin E1) of platelet aggregation. Platelets from control subjects and patients with Raynaud's syndrome were more resistant to prostaglandin inhibitors when reactions were carried out at 27 degrees C rather than at 37 degrees C. Patients with Raynaud's syndrome also had significantly increased plasma concentrations of beta-thromboglobulin, fibrinogen, and circulating platelet aggregates. In an attempt to elicit local platelet responses, the forearms of control subjects and patients with Raynaud's syndrome were cooled in water tanks and platelet function tests performed before and after cooling. No significant difference in the results was observed. The potential role of platelets in the pathogenesis of Raynaud's syndrome is discussed.

Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma).

Serial venom antigen levels were measured by enzyme-linked immunosorbent assay (ELISA) in 46 patients with systemic envenoming by the Malayan pit viper (Calloselasma rhodostoma), a major cause of snake bite in Southeast Asia. The principal effects of the venom are defibrination, hemorrhage and local tissue necrosis. Admission venom levels, which varied between 0 and 595 ng/ml, correlated with the incidence of spontaneous systemic bleeding, blood incoagulability and concentrations of plasma fibrinogen and serum fibrin degradation products. The presence or absence of nonclotting blood also correlated with the time elapsed between the bite and hospital admission. The development of nonclotting blood may be delayed by up to 72 hr after the bite even though circulating venom and raised FDP may be detected at presentation. This is probably explained by a temporary equilibrium between synthesis and consumption of fibrinogen. Venom antigenemia recurred in 12 patients (26%) suggesting continuous absorption of venom from the wound or saturation of extravascular binding sites. Admission venom levels also correlated with the extent of local swelling and the occurrence of tissue necrosis at the site of the bite. Venom was detected in 87% of wound aspirates and 88% of urine specimens taken on admission. Tourniquets, of the type used in rural Thailand, did not delay the absorption of venom into the circulation.

Randomized comparative trial of three monospecific antivenoms for bites by the Malayan pit viper (Calloselasma rhodostoma) in southern Thailand: clinical and laboratory correlations.

Three monospecific antivenoms for Malayan pit viper (MPV) (Calloselasma rhodostoma) were compared in Southern Thailand, where this species is the most common cause of snake bite morbidity. Forty-six patients with proved MPV bites and incoagulable blood, indicating systemic envenoming, were randomly allocated for treatment with Thai Red Cross (TRC), Thai Government Pharmaceutical Organization (GPO), or Twyford Pharmaceutical monospecific antivenoms. Both GPO and Twyford antivenoms produced rapid and permanent restoration of blood coagulability, but TRC antivenom failed in 2/15 cases. Patients in the GPO group showed the greatest increase in plasma fibrinogen concentration during the first 24 hr and had fewer early anaphylactic reactions (6/15) compared with Twyford 8/16 and with TRC 13/15. Pyrogenic reactions occurred more frequently after TRC antivenom (8/15) than GPO (1/15) or Twyford (0/16). Patients requiring more than one dose of antivenom were identifiably more severely envenomed on admission. In an accompanying laboratory study the antivenoms were assessed in rodents using five WHO standard tests of neutralizing activity. Compared with the other two antivenoms TRC was significantly inferior in anti-lethal potency, GPO was superior in anti-hemorrhagic and anti-necrotic potency and Twyford was superior in anti-procoagulant and anti-defibrinogenating potency. The clinical efficacy of these antivenoms against local necrosis remains equivocal. GPO and Twyford antivenoms are recommended for the treatment of systemic envenoming by MPV in an initial dose of 5 ampoules.

Neurotoxicity, anticoagulant activity and evidence of rhabdomyolysis in patients bitten by death adders (Acanthophis sp.) in southern Papua New Guinea.

Thirty-two patients with enzyme-immunoassay-proven death adder (Acanthophis sp.) bites were studied in Port Moresby, Papua New Guinea. Eighteen were envenomed; local signs were rare and none had incoagulable blood, but all except one had signs of neurotoxicity. Five (27.7%) envenomed patients required intubation and ventilation. One patient developed renal failure, previously undescribed following death adder bites. Laboratory investigations showed mild prolongation of prothrombin and partial thromboplastin times in some patients. In vitro studies showed that the venom contains anticoagulant activity, but does not cause fibrinogenolysis. In contrast to taipan envenoming, neurotoxicity did not progress after antivenom administration, and there was reversal of neurotoxicity, evident within 6 h, in three severely envenomed patients treated less than 12 h after the bite. One patient treated with antivenom and anticholinesterases had the most dramatic response to treatment; the optimum management of bites by this species may include prompt treatment with both antivenom and anticholinesterases in addition to effective first aid.

Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment.

Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of D-penicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease.

In vitro studies of Russell's viper venom on blood coagulation and fibrinolysis.

The effect of Russell's viper venom (RVV) on clot formation and lysis and the effect thereon of specific antivenom for Russell's viper venom, were studied in vitro. RVV had fibrinogenolytic activity, but only at a concentration greatly in excess of that likely to be achieved in vivo after Russell's viper bite. Similarly, RVV did not directly activate the fibrinolytic system in vitro, even at very high concentrations (10,000 micrograms/ml). Furthermore, 40 ml of antivenom (ASV) was found to be sufficient to neutralise the average amount of venom (60 mg) injected by a snake, but insufficient to neutralise amounts in excess of 75 mg.

Development and evaluation of ELISAs for factor XIIIA and XIIIB subunits in plasma.

Severe, congenital deficiency of factor XIII is extremely rare. However, a moderate reduction in the plasma level of the functional subunit (factor XIIIA) and also to a lesser extent of the carrier subunit (factor XIIIB), and a decrease in the XIIIA:B subunit ratio, have recently been reported in patients with the inflammatory bowel disorder Crohn's disease, particularly during clinical relapse. In order to accurately monitor patients, sensitive, reliable assays for the two subunits of factor XIII are required. We report here the development and validation of ELISAs for these components. The assays are identical except in respect of the specificity of the polyclonal antiserum used as starting material, both of which are commercially available. The antisera are purified by n-octanoic acid precipitation and portions of these purified immunoglobulins are used as coating antibodies. The remaining portions are biotinylated and used with streptavidin and horse-radish peroxidase as tracer antibodies. A normal range (n = 24) was established for factor XIIIA (mean 95 range 60-130 U/dl) and for factor XIIIB (mean 99 range 60-130 U/dl). There were no significant differences between the ELISA and electroimmunodiffusion assays either for factor XIIIA (means +/- 1 standard deviation 95 +/- 15.9 and 89 +/- 22.7 respectively) or for factor XIIIB (99 +/- 18.3 and 106 +/- 23.4 respectively). These assays have been in routine use for six months, during which time two further antisera purifications and biotinylations have been carried out without significant problems of reproducibility or stability.

Platelet aggregation in myotonia.

Platelet function tests were carried out on 21 patients with myotonic dystrophy (MyD) and 7 patients with myotonia congenita (MC) together with 22 healthy subjects. Compared to the controls, the MyD and MC patients showed significantly greater sensitivity to the calcium ionophore A23187 but not to adenosine diphosphate, collagen, ristocetin or adrenaline. In addition, with the MyD patients, a significantly higher concentration of chlorpromazine and lignocaine was required to inhibit aggregation induced by a standard dose of adrenaline. A similar trend was noted with the MC patients but because of the small numbers tested, statistical analysis was not possible. Plasma levels of betathromboglobulin were also significantly higher in MyD patients than in controls. Platelet adenine nucleotide levels were within the normal range in both groups of patients and were not significantly different from those in controls. These preliminary results support the view that there is a defect in calcium metabolism in myotonic dystrophy and suggest that it may be possible to use the blood platelet as a model with which to carry out further studies in this disorder.