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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.

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.

Antibodies against factor VIII collected from six patients were studied for their effect on factor-VIII coagulant activity, Willebrand factor activity (WF) and factor-VIII-related antigen. The results demonstrate differences between individual antibodies; some acting primarily as anti factor VIII, others inhibiting both after VIII and WF. Differences in effect between plasma and serum indicate that the process of coagulation alters the interaction of these antibodies.

SummaryPlasmas from ten patients with a lupus or lupus-like inhibitor were investigated. In each case the partial thromboplastin time with kaolin (PTTK) was prolonged and failed to correct on the addition of an equal volume of normal plasma. Activated control platelets corrected the inhibitory effect in the PTTK or thrombin generation time (TGT) in every instance. Activated autologous platelets were as effective as control platelets and may thus explain why bleeding is rarely associated with the lupus inhibitor. Experiments using platelets or plasma from patients congenitally deficient in a single clotting factor or normal washed platelets resuspended in deficient plasma indicated that the inhibitor bypassing activity is platelet and not plasma derived. Platelet fractionation studies suggested that this activity is localised at the platelet membrane.

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.

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.

In the presence of epsilon aminocaproic acid (EACA) thrombin generation in recalcified platelet rich plasma (PRP) was markedly stimulated, as measured by the cleavage of the synthetic substrate S2238. However, thrombin activity remaining after 30 minutes incubation was reduced when compared with control values. The residual activity was shown to be hirudin insensitive and to be associated with a species of higher molecular weight than free thrombin. These results suggested an inhibition of thrombin binding to the antithrombin, alpha 2-macroglobulin (alpha 2M). Preincubation of PRP with EACA reduced the concentration at which EACA elicited its dual effects. Similar results were obtained with the alpha 2M inhibitor, hydrazine. These experiments indicated that alpha 2M may play a more important role in regulating thrombin generation than has been previously recognized.

Previous authors have noted that discrepancies occur for factor VIII-related antigen (VIII-Ag) levels when different techniques are used for its measurement. However no systematic study has been undertaken to identify the causes for these discrepancies. Factor VIII-related antigen (VIII-Ag) has been quantitated by either electroimmunoassay (EIA), solid or liquid phase radioimmunometric assay (S-IRMA or L-IRMA respectively). In general a close correlation existed between levels of VIII-Ag measured by these techniques. Values for VIII-Ag in serum, cryosupernatant and the plasma of haemophiliacs who have developed antibodies to factor VIII(FVIII) were found to be lower when measured by S-IRMA than by EIA; the L-IRMA gave intermediate values. Wherever a lack of correlation existed between the assays the VIII-Ag was found to have an increased electrophoretic mobility on radio 2-dimensional immunoelectrophoresis (radio 2-DIEP). Measurement of VIII-Ag of column fractions from a purification of FVIII concentrate confirmed that the S-IRMA failed to detect small VIII-Ag forms. It could be demonstrated that the antibody used for IRMA showed selection of the antibody towards large VIII-Ag forms. This selection together with the ‘two antibody sandwich’ technique used in S-IRMA may explain loss of reactivity towards the small VIII-Ag forms of FVIII.

A simple method for measuring the control of blood loss in thrombocytopenic rats and mice is described. The administration of lyophilized platelets, brain phospholipid extract and soya bean extract failed to correct the blood loss in the thrombocytopenic animal. The results obtained with this method have failed to support the effectiveness of "platelet substitutes" in in vivo systems and reaffirm the requisites of viability and recirculation for platelet transfusions.

Electron microscopic examination of splenic tissue obtained from patients with a variety of hematologic disorders has shown ultrastructural evidence for platelet breakdown within splenic histiocytes in seven out of eight patients with idiopathic thrombocytopenic purpura (I. T. P.). The morphologic characteristics of this intracellular digestion have been described.

Viscous metamorphosis of platelets has been studied with the light microscope, and ultra-thin sections have been prepared at progressive stages for examination in the electron microscope. The phase contrast light microscope reveals rapid aggregation and distortion of platelets and gives the impression of their fusion into structureless aggregates during viscous metamorphosis. Sectioned material collected during viscous metamorphosis of platelets and examined in the electron microscope reveals a remarkable degree of retention of structure in a majority of the platelets. All become deficient in granules and devoid of vesicular spaces, but most retain intact cell membranes, and the structureless masses seen with the light microscope are found to consist of densely aggregated platelets. Fusion and complete loss of identity occurs in the minority. The retracted clot was found to contain densely aggregated, distorted and elongated platelets, empty of granules and intimately related to fibrin particles.

Summary1. The antibotic, Ristocetin, causes precipitation of fibrinogen from platelet poor plasma, as well as platelet aggregation.2. The precise mechanism of Ristocetin’s effect on platelets has not been elucidated, but it has been shown to initiate ADP release, which may contribute in part to its aggregating ability.3. Ristocetin has been shown to produce aggregation in platelet rich plasma from thrombasthenic patients.4. Three patients with Von Willebrand’s disease were examined. In 2, Ristocetin caused no platelet aggregation whatsoever, whilst the third aggregated normally. It is suggested, on this basis, that Von Willebrand’s disease may be subdivided into two types and that Ristocetin could prove to be a valuable technique for further study of this group of disorders.

Summary1. A correlation between platelet aggregation and the plasma enzyme(s) ability to degrade Adenosine Diphosphate (ADP) has been confirmed.2. This plasma activity has been shown to be reduced in 6 patients with uraemia in whom platelet aggregation was demonstrably impaired but not in two whose platelet function was normal. The incorporation of 14C labelled ADP-8-14C was also only reduced in uraemic patients with abnormal platelet aggregation.3. These findings are discussed with particular reference to possible implication in mechanism involved in ADP aggregation of platelets.

Surgically removed spleens from 14 subjects were perfused to determine their platelet content. Some patients had Cr51 labeled platelets prior to surgery, and radioactive platelet recoveries were also obtained. A sizable pool of splenic platelets has been demonstrated, which seems dependent on splenic size only. Increase in size of this pool in splenomegaly (and not suppression of thrombopoiesis) may be the sole reason for thrombocytopenia. A dynamic exchange between splenic and circulating platelets appears to be present. Awareness of the existence of a splenic platelet pool is important in considering variation in platelet survival and other platelet dynamic studies, especially in the presence of splenomegaly.

Summary. Morphological features of the peripheral blood of the ancestral elasmo-branch Heterodontus portusjacksoni, a shark species, are described. The red cell is nucleated and contains many membrane-lined vacuoles which are derived from mitochondria. The granulocytic series resembles that of the mammals in appearance and in some phagocytic properties. The thrombocytes possess a number of similarities to mammalian platelets, but exhibit many differences. They aggregate readily, but this is temperature reversible and is independent of thrombin and ADP. They show a remarkable affinity for latex particles. Unlike many other species, fibrin formation and thrombocyte aggregation are apparently independent phenomena occurring in parallel rather than in series. Tritiated thymidine studies and the observation of mitotic figures in the peripheral blood suggest that at least part of the red cell and thrombocyte proliferation occurs in the circulating blood.

Three patients with drug-induced hypoplastic anemia terminating 2 to 6 years after presentation with erythroleukemia are described. All were treated for prolonged periods with androgen and corticosteroid and two of the patients showed apparent dependence on this therapy for optimal hematologic status. The leukemic phase was heralded by loss of this dependence and development of sideroblastic dyserythropoiesis with progression to bizarre erythroid hyperplasia and fatal cytopenia. The exact relationship between androgen and corticosteroid therapy and the erythroleukemia remains speculative.

Thrombotic thrombocytopenic purpura, an acute clinical disorder of uncertain aetiology, has previously been associated with almost 100% mortality. Dramatic response to exchange transfusion has been reported, and there is some evidence that a circulating immune complex may be involved in the pathogenesis. Therefore, it was decided to employ large-volume plasma exchange by means of a continuous-flow cell separator in the management of five patients with this disease. Three patients showed a dramatic response, which was manifested by rapid correction of the neurological deficit and of the abnormalities in laboratory findings. We conclude that the thrombotic thrombocytopenic syndrome should be regarded as an indication for emergency plasma exchange.

Platelet associated IgG (PA-IgG), serum IgG and immune complexes (IC) were measured and correlated with the degree of thrombocytopenia in 32 patients with chronic active hepatitis (CAH), classified as of autoimmune type in 24 and cryptogenic in eight. Of the 32 patients, 24 had raised PA-IgG and 18 of these were thrombocytopenic (greater than 150 X 10(9) platelets/1); in only two thrombocytopenic patients was the level of PA-IgG not increased. Platelet counts showed a significant inverse logarithmic correlation with PA-IgG levels (r = 0.456, P less than 0.01). Twelve patients had raised levels in serum of ICs, 11 of these also had raised PA-IgG and 10 of these 11 were thrombocytopenic. Serum IgG was raised in 10 patients and correlated with PA-IgG (r = 0.489, P less than 0.01). Of the 24 patients with autoimmune CAH, 13 had low platelet counts and 10 had raised levels of PA-IgG whereas of the eight patients with cryptogenic CAH, seven had raised PA-IgG and all were thrombocytopenic. These results for patients with CAH show that raised levels of circulating ICs are associated both with thrombocytopenia and with raised PA-IgG, and support the hypothesis that serum IgG and/or ICs may influence amounts of PA-IgG.

Normal human factor VIII (FVIII) is a high molecular weight glycoprotein expressing two measurable biological activities, FVIII procoagulant activity (VIII:C) and von Willebrand factor activity (vWf). The corresponding antigenic moieties are referred to respectively as FVIII coagulation antigen (VIII:CAg) and FVIII related antigen (VIII: RAg). Since VIII:C is extremely labile measurement of VIII: CAg is of importance for pre-natal diagnosis of haemophilia where VIII:C is reduced and von Willebrand’s disease (vWd) where all measurable parameters of FVIII are reduced.Current immunoradiometric assays for VIII:CAg depend on both the availability of high titre human antibodies directed against VIII:C and the possibility of preparing highly specific 125I-IgG from human antiserumWe have developed a solid phase immunoradiometric assay for VIII:CAg. FVIII in a test sample binds to human anti- VIII:C IgG immobilised on the surface of a polystyrene tube. The bound FVIII is detected using rabbit 125I-anti human FVIII-IgG. The standard curve was linear for dilutions of normal pooled plasma (NPP) of 1/5 to 1/500. The lower limit of the assay was 0.002U/ml of NPP. Using this assay the concentration of VIII:CAg correlated well with the VIII:C levels in the plasma from normal individuals (r=0.84, n=15). Homozygote vWd patients had undetectable levels of VIII:CAg, VIII:C, VIII:RAg or vWf activities. Patients with haemophilia A with less than O.OlU/ml VIII:C could be divided into two groups on the basis of the VIII:CAg assay, CRM and CRMf. Markedly reduced levels of VIII:CAg were detected in serum.The advantage of this assay over the previously available assays for VIII:CAg are that smaller volumes of high titre human anti VIII IgG are required and the time consuming step of preparing 125I-human anti VIII-IgG is eliminated. The rabbit 125I-IgG specific for human VIII is the same as used in routine immunoradiometric assays for FVIII:RAg

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.

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.

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.

In three patients with thrombocytosis secondary to myeloproliferative disorders, disabling intermittent lower extremity pain occurred in the absence of peripheral vascular insufficiency. In all patients, circulating platelet aggregates were detected. Antiplatelet therapy resulted in prompt relief of pain and the disappearance of platelet aggregates. These findings suggest that abnormal platelet function with the formation of platelet plugs in the microcirculation could be responsible for the observed symptoms and response to treatment. The value of the determination of circulating platelet aggregates in the management of this syndrome is highlighted.

Four patients with warm type autoimmune hemolytic anemia who failed to respond to steroid therapy received high dose intravenous gammaglobulin (0.2-0.4 g/kg daily) for five days. In one patient hemolysis occurred in association with non-Hodgkin's lymphoma and in the others the cause of the hemolysis was not established; two patients had prior splenectomy. A response to gammaglobulin therapy, demonstrable by a rise in or stabilisation of hemoglobin levels, a decrease in elevated serum bilirubin and lactate dehydrogenase levels, or prolongation of a shortened red cell (51Cr) survival, was observed in three patients. In two of these patients the effect of gammaglobulin therapy was temporary but allowed for splenectomy to proceed in one patient, without blood transfusion. The third patient continued to improve after cessation of gammaglobulin treatment. These findings suggest that high dose intravenous gammaglobulin may temporarily ameliorate hemolysis in some individuals with warm type immune hemolytic anemia, and may be a useful adjunct to steroids immediately before splenectomy.

A proteolytic product of vWf termed fast migrating protein (vWf:FMP) has been identified using crossed immunoelectrophoresis (CIE) in normal serum and in the plasma from patients with disseminated intravascular coagulation (DIC). A fragment of vWf antigen (vWf:Ag) migrating to a similar position on CIE as vWf:FMP results from digestion of vWf:Ag with polymorphonuclear cells (PMNC). Since parallels exist between the conditions for generation of vWf:FMP by PMNC and elastase release by these cells the effect of purified elastase from porcine pancrease on vWf was investigated.VWf was purified from plasma using polyethylene glycol, ammonium sulphate and zinc acetate precipitation, high speed centrifugation and elution from column of Sepharose 4B-CL. A fraction rich in vWf was radiolabelled with 125-Iodine to spike the purified preparation of vWf in order to increase the sensitivity of the protein detecting systems.A mixture of radiolabelled and non-labelled purified vWf was incubated with elastase at concentrations ranging from 2.5 to 40 U/ml for periods of 0-48 hours. Modifications of the structure were assessed by SDS-agarose multimeric analysis, SDS-polyacrylamide electrophoresis and CIE. Alterations of function were quantitated by antigen levels, ristocetin (RCof) and botrocetin (BCoF) cofactor assays and a binding assay to fixed washed platelets in the presence of ristocetin or botrocetin.These investigations show, 1. all but the highest molecular weight multimers of vWf are present when elastase has cleaved vWf such that no intact 240K subunit is present. 2. an intact 240K subunit is not essential for RCoF and BCoF activity or for ristocetin or botrocetin induced binding to platelets.

Platelet-associated IgG (PA IgG) of washed platelets of 20 normal controls and 45 thrombocytopenic patients was measured by radioimmunoassay and related to total platelet protein content. Mean values (+/- 2 SD) for 20 normals were 4.4 +/- 3.5 fg IgG per platelet (fg IgG/pl), 2.1 +/= 1.0 pg protein per platelet (pg protein/pl) and a ratio of IgG per g platelet protein of 2.1 +/- 1.8 mg IgG/g protein. Patients could be divided into two groups: Group I (31) with normal IgG/g protein ratios and either normal (16) or raised (15) fg IgG/pl, and Group II (14) with both fg IgG/pl and IgG/g protein raised. Of the 21 patients with idiopathic thrombocytopenia (ITP), 13 were in Group I (seven with raised and six with normal fg IgG/pl), and eight in Group II. There was significant correlation between PA IgG (fg IgG/pl) and platelet protein for the entire Group I (r = 0.95, P less than 0.001). The ITP patients in this group showed a similar correlation (r = 0.77, P less than 0.001), suggesting that raised PA IgG levels in Group I patients may be a result of protein-rich platelets. 60-90% of PA IgG was found in the soluble fraction of lysates of platelets from both normals and patients. Thus, it is either non-specifically associated with platelets or is antibody directed towards easily solubilized platelet antigens. Nine thrombocytopenic patients without immune disorders had raised PA IgG. These results question the assumption that raised PA IgG always indicates specific antiplatelet antibody.

The effect of chloramphenicol in short term in-vitro bone marrow cultures was studied. There was a striking reduction in the incorporation of tritiated thymidine into DNA in bone marrow cultures with abnormal proliferative properties as compared with normal tissue. A 50% reduction in DNA thymidine incorporation in leukaemia marrow was also obtained with in-vitro chloramphenicol concentrations which in contrast had little or no effect in normal tissue. These in-vitro levels of the antibiotic can be readily achieved in vivo. An in-vivo study confirmed the ability of chloramphenicol to reduce the white cell and blast count in a patient with chronic myeloid leukaemia in blastic transformation.

The initial interaction of polystyrene latex particles with platelets has been studied by means of their agglutination with platelets measured nephelometrically .2 to 10 min. after addition of latex particles to citrated PRP at 37°C slow agglutination occurs. When latex is preincubated with PPP for 5 to 40 mins at 37°C, sedimented and resuspended in buffer it is “activated” and produces an immediate and rapid agglutination when added to PRP. This “activation” like activation of zymosan particles (Z) occurs in plasma deficient in Factor VIII and/or von Willebrand factor, is not prevented by hirudin, and does not occur in plasma heated at 56°C for 10 min or in recalcified PPP; addition of fibrinogen restores activity to these treated plasmas. Unlike Z activation, it is not inhibited by heparin, EDTA, or destruction of Complement. Activation can be mimicked by preincubation with fibrinogen alone, but not with IgG or albumin. In PRP, in contrast to washed platelets, latex preincubated with IgG caused less agglutination than when pretreated with fibrinogen or buffer. Agglutination by activated latex in PRP, unlike aggregation by activated Z and other aggregating agents, causes only slight 14C-serotonin release and is not inhibited by aspirin, indomethacin or adenosine,but, like the reaction with Z, is inhibited by EDTA, chlorpromazine, cocaine-HCl, N-ethylmaleimide and iodoacetate. Platelets from patients with thrombasthenia show a defective response to latex activated with either normal PPP or fibrinogen. Particles of inulin which activate C in the same way as Z, but which absorb much less fibrinogen, cause no response of either normal PRP or washed platelets. Thus normal platelets, but not those from thrombasthenics, appears to possess a receptor for fibrinogen which is important in their interaction with particulate material.