As time goes by: reflections on 40 years of academic medicine and biomedical research.

Professor Firkin of the Department of Medicine at Melbourne's Monash University says that, despite considerable advances, many unanswered questions remain in modern medicine. The bright medical minds that may answer these questions are being lost to the cause because of rigid postgraduate training schemes and inadequate remuneration.

von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.

Studies of natural anticoagulant proteins and anticardiolipin antibodies in patients with the lupus anticoagulant.

Components of the natural anticoagulant system (NAS) and anticardiolipin antibodies were examined in 21 patients with lupus anticoagulant (LA), 13 of whom had past histories of thrombotic episodes. No relationship could be shown between the antigenic levels of protein C and S (PC, PS) and a history of thrombosis. Inhibition of the anticoagulant activity of activated protein C (APC) was observed using plasma from 20/21 patients when phospholipid vesicles were used as the surface for the coagulation reaction. This effect was not affected by the addition of PS. When platelet membranes were employed only 2/21 patients demonstrated inhibition of APC. Under the latter condition, PS functional activity was inhibited in 7/21 patients, six of whom had a past history of thrombosis. Reduced antithrombin III or heparin cofactor II levels were observed in a total of 4/21 patients and may have contributed to the development of thrombosis in three of these patients. Antibodies specifically directed against these proteins were not detected suggesting the possibility of an associated constitutional deficiency. Anticardiolipin antibodies, though elevated in 17/21 patients, did not serve as a useful marker for an increased risk of thrombosis, and the level did not correlate with inhibition of the activity of APC or PS. We conclude that the mechanism of thrombosis in patients with LA is multi-factorial. A subset of patients in whom LA specifically inhibits PS function may represent patients who are at significant risk from thrombosis.

Platelet aggregating IgG antibody to platelet surface glycoproteins associated with thrombosis and thrombocytopenia.

Previously described platelet-aggregating antibodies associated with thrombosis and thrombocytopenia required heparin for their in vivo and in vitro expression. We have observed a patient with thrombosis who became thrombocytopenic during heparin treatment, but who suffered further thrombotic events and continued thrombocytopenia for 3 months after heparin withdrawal. The patient's plasma contained a potent platelet aggregating factor reactive with both his own and normal platelets in the absence of heparin. It also caused [14C]serotonin secretion from labelled platelets from normal donors and patients with either Glanzmann's thrombasthenia or Bernard-Soulier syndrome. This factor was an IgG and was neutralized by antibody specific for IgG lambda light chains. While the patient was thrombocytopenic an IgG paraprotein with lambda light chains was detected by isoelectrofocussing. After corticosteroid treatment it disappeared and the patient recovered. The active, but not the recovery serum contained IgG which immunoprecipitated a glycoprotein with characteristics of Glycoprotein IV from platelets labelled with Na[3H]BH4/periodate. Thus platelet-aggregating IgG antibodies with direct specificity for platelet surface glycoproteins may be associated with thrombosis/thrombocytopenia.

Impaired fibrinolysis in patients with thrombotic or haemostatic defects.

In a retrospective study of the 47 patients examined for thrombotic defects, 34 had prolonged euglobulin lysis times (ELT) with 27 of these also having elevated tissue-plasminogen activator inhibitor (t-PAI) activity. Twenty three of the 57 patients examined for possible bleeding problems had prolonged ELT with 15 of this group also having raised levels of t-PAI. There was a statistically greater (P less than 0.005) incidence of elevated t-PAI in the thrombotic group. The incidence of 40% patients with prolonged ELT in the haemostasis group was surprising and perhaps represents some compensatory mechanism to combat bleeding. Thus in vitro tests showed that a reduced capacity for fibrinolysis was present in diametrically opposed groups.

Comparison of laboratory tests used for identification of the lupus anticoagulant.

A comparison of the sensitivities of the ten most commonly used tests for the identification of the lupus anticoagulant (LA) and the lupus cofactor phenomenon was undertaken on 18 patients. All investigations, except the cardiolipin-antibody ELISA assay, were carried out using patient's plasma alone followed by a 1:1 mix with control plasma. Dilution studies (1:3, 1:6, 1:9--patient:control) were also carried out. The kaolin clotting time (KCT) was the only test positive in all patients at all dilutions, while the dilute activated partial thromboplastin time with kaolin (Dil-APTT) registered 17 of 18 positive at all dilutions. Both the dilute Russell viper venom time (Dil-RVVT) and the tissue thromboplastin inhibition time (TTI) (1/500 thromboplastin) identified the LA in 17 of 18 patients on initial testing but were less sensitive in the dilution studies. The KCT is not a suitable test for routine laboratory use, as it requires an individual filtration step. Therefore a combination of either the Dil-APTT or Dil-RVVT together with the TTI (1/500 dilution thromboplastin) is recommended for routine LA screening, as all patients with LA in this study were identified using these easily automated tests. The lupus cofactor phenomenon was most frequently demonstrated using the Dil-APTT.

Differing susceptibilities of platelets from normal individuals and patients with von Willebrand's disease to attack by quinine- and quinidine-dependent antiplatelet antibodies.

Reactivity of quinine- and quinidine-dependent antiplatelet antibodies has been compared in platelet-rich-plasma (PRP) from normal donors and from patients with von Willebrand's disease (vWd). One quinine-dependent antibody (Q.Ab) caused platelet aggregation and [14C] serotonin release with only 7 of 12 normal donors, while another Q.Ab and a quinidine-dependent antibody (Qd.Ab) caused aggregation and release with all 12. Drug-dependent IgG binding and PF 3 availability induced by the antibodies were, however, comparable in all donors. Differences in responsiveness were associated with platelets and not plasma. vWd platelets showed normal drug-dependent IgG binding, but decreased aggregation and serotonin release to most drug-dependent antibodies. Responsiveness was not restored by purified vWf:Ag, but, in one case, was corrected by normal plasma or cryoprecipitate. Drug-dependent binding of the Q.Ab which caused variable responsiveness in normals was to the same platelet antigens (GPIb and GPIIIa) in both normal and vWd platelets and did not require plasma components. Reduced PF 3 availability was seen with some antibodies in some vWd patients. Plasma from two of these patients inhibited aggregation of normal platelets to Q.Ab and one of these inhibited aggregation to ADP. Antiplatelet antibodies were detected in these two plasmas by ELISA. Thus some Q.Ab produce different responses with platelets from different donors. In vWd, reduced responsiveness to Q.Ab and Qd.Ab may result from production of inhibitory antiplatelet antibodies.

Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of glycoproteins Ib, IIb, IIIa, and IX.

The molecular nature of platelet receptors for quinine- and quinidine-dependent antiplatelet antibodies (Q.Ab and Qd.Ab) was studied by immunoblotting. One Q.Ab caused quinine-dependent IgG binding to platelet proteins with molecular weights (mol wts) of 174 Kd and 93 Kd and another to only a 93-Kd protein. A third Q.Ab caused binding to 174-, 140-, 93-, and 57-Kd proteins, while a fourth Q.Ab and a Qd.Ab caused IgG binding to 174- and 18-Kd proteins. Using platelets from patients with Glanzmann's thrombasthenia or Bernard Soulier syndrome and purified GPIIIa, these proteins were shown to be GPIb, GPIIb, GPIIIa, GPIX, and an unidentified 57-Kd protein missing in Bernard Soulier syndrome. Binding to the 93-Kd protein was independent of the PIA1 antigen. Absorption of one Q.Ab with Glanzmann's thrombasthenia platelets revealed different populations of antibodies with different specificities within the one patient. Thus Q.Ab and Qd.Ab are heterogeneous and may be directed toward different epitopes on major platelet glycoproteins.

Management of refractory idiopathic thrombocytopenia.

In refractory thrombocytopenia one should first evaluate whether the therapeutic approach has more risks to the patient than no treatment at all. The patient may remain relatively asymptomatic and the only incommoding circumstances be cosmetic. Coincidental medical problems such as hypertension and peptic ulceration are particularly worrying in the patient with ITP. Very occasionally a cerebral haemorrhage may occur without any obvious predisposing cause. In most instances, however, refractory thrombocytopenia is a benign condition which rarely directly causes the death of the patient. Since many of the remedies advocated for this problem have significant side effects these must always be carefully balanced and fully discussed with the patient before their introduction.

Comparison of two dimensional immunoelectrophoresis and multimer analysis in the study of von Willebrand factor.

To assess the validity of the multimeric analysis of von Willebrand factor (vWf), this technique was compared with two dimensional immunoelectrophoresis using samples of purified vWf obtained by gel filtration and plasma samples from a patient with severe von Willebrands disease who was receiving prophylaxis with cryoprecipitate. It is concluded that except for specialised clinical and research purposes, two dimensional immunoelectrophoresis provides a clear picture of the multimeric composition of the vWf molecule, and that this is sufficient for routine research and clinical use, without having to confirm the data by multimeric analysis.

Effects of nicotine on platelet function.

In non-smokers, only high concentrations of nicotine (10 mM) caused platelet aggregation in platelet-rich plasma and release of 5-hydroxytryptamine (5-HT). Both responses to ADP and 5-HT were enhanced at 1 and 10 mM nicotine while they were inhibited to collagen, ristocetin, adrenaline and arachidonic acid. Lower concentrations of nicotine had no effect with any agent other than 5-HT, with which a variable enhancement of 5-HT-induced aggregation was observed. Uptake of 14C-5-HT was inhibited by nicotine at 100 microM or higher while platelet factor 3 availability was unaffected. Thus it is unlikely that direct effects of nicotine on platelets are responsible for smoking-related changes in platelet reactivity.

Lupus anticoagulant in women with multiple spontaneous miscarriage.

A link between the presence of the lupus anticoagulant (LA) and recurrent spontaneous miscarriage exists; however, the incidence of the LA in patients with unexplained recurrent miscarriage is unknown. We have therefore investigated the incidence of the LA in women with recurrent miscarriage but without evidence of systemic lupus erythematosus or other syndromes known to predispose towards spontaneous abortion. Plasma from 29 such women was obtained and tested in the activated partial thromboplastin time (APTT), testing for correction with normal plasma if prolonged and in the dilute simplastin time test (DSTT), again mixing with normal plasma if prolonged. Using a prolongation in either/or both tests which was not corrected by mixing with normal plasma as criteria for the presence of the LA, 14 (48%) patients showed evidence for the presence of the LA. Substitution of platelet-rich plasma for phospholipid and plasma in the APTT corrected the prolonged clotting time in every patient (9/29) in which it was prolonged, further substantiating the presence of the LA. We suggest that the presence of the LA should be investigated in any patient with unexplained recurrent miscarriage.

Analysis of immunoglobulins that bind to platelets from serum of patients with immune thrombocytopenia: molecular weight distribution.

The nature of platelet- bindable immunoglobulins (PB-Ig) in serum has been investigated. PB-IgG, -A and -M were measured by an ELISA using platelets coated on microtitre plates. This assay detected alloantibodies at high serum dilutions. In 32 patients with idiopathic thrombocytopenic purpura (ITP) or systemic lupus erythematosus (SLE) raised levels of at least one PB-Ig class were found in 18. To distinguish binding due to immune complexes, the molecular weight of PB-IgG was studied by gel filtration on Sepharose 4B. In sera from patients with ITP and SLE, PB-IgG with Mr of primarily 150 Kd was observed, compatible with monomeric IgG antiplatelet antibodies. Levels of PB-IgG in serum were not related to total serum IgG. In sera from the patients with SLE and some with ITP (most of whom had several of the features of SLE), PB-IgG with Mr of 200 Kd - greater than 1000 Kd was seen. In heat-aggregated preparations of normal IgG, PB-IgG with Mr up to 1000 Kd was also found. Rabbit IgG was able to block PB-IgG in fractions of high molecular weight in purified normal IgG, heat-aggregated normal IgG and in patient serum, but had no effect on the 150 Kd peak. In whole serum from patients who had high molecular weight PB-IgG, the inhibitory effects of rabbit IgG were much less than in isolated high molecular weight column fractions. Thus although the majority of PB-IgG is monomeric antiplatelet antibody, some PB-IgG with higher molecular weight, characteristic of immune complexes, occurs in sera of some patients with autoimmune thrombocytopenia and it makes a small contribution to PB-IgG levels measured in whole serum.