G Purdy

Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.

Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 ± 2.0 mg versus 17.1 ± 1.4 mg, P 1 (I) procollagen and CTGF mRNA levels (3.0 ± 0.4-fold and 6.3 ± 0.4-fold respectively, ( P P 1 (I) procollagen and CTGF mRNA levels by 50% and 35%, respectively ( P

Microvesicles in haemoglobinopathies offer insights into mechanisms of hypercoagulability, haemolysis and the effects of therapy.

Levels of circulating red blood cell (RBC)‐derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6‐fold greater in SCA and 4‐fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40–50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)‐treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

The use of an anti-β2-glycoprotein-I assay for discrimination between anticardiolipin antibodies associated with infection and increased risk of thrombosis

Summary. Antiphospholipid antibodies (aPAs), occurring in association with infection, are not generally associated with an increased risk of thrombosis. Anticardiolipin antibodies (aCL) from patients with infection, unlike those from patients with SLE, do not have the β2GPI cofactor requirements. Antibodies to β2GPI (αβ2GPI) are more closely associated with a previous history of thrombosis than aCL in patients with SLE. In the present study we have investigated the reactivity of the αβ2GPI assay for aPAs associated with infection. Serum from 114 patients with infections including syphilis (n= 11), tuberculosis (n= 63) and Klebsiella (n=42) were assayed for αβ2GPI and aCL antibodies. The incidence of aCL in serum of patients with tuberculosis, Klebsiella infection and syphilis was 6.0%. 5.0% and 64.0%. respectively, but all patients were negative for αβ2GPI. These results indicate that the αβ2GPI assay is negative in patients with transiently positive aCL assays associated with infection.

Platelet degranulation and monocyte-platelet complex formation are increased in the acute and convalescent phases after ischaemic stroke or transient ischaemic attack.

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte–platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P‐, CD63‐, and PAC1‐binding, and the percentages of leucocyte–platelet complexes in acute (1–27 d, n = 79) and convalescent (79–725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P‐selectin, soluble E‐selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte–platelet complexes were higher in both acute and convalescent CVD patients than controls (P ≤ 0·02). The mean white cell count and mean VWF:Ag levels were significantly elevated in the acute and convalescent phases after ischaemic stroke or TIA (P ≤ 0·02). Otherwise, there was no significant increase in any other marker of platelet or endothelial activation in CVD patients. There was a positive correlation between the percentage expression of CD62P and the percentages of both neutrophil–platelet and monocyte–platelet complexes in the acute phase, and the percentages of all leucocyte–platelet complexes in the convalescent phase after ischaemic CVD. This study provides evidence for ongoing excessive platelet and/or endothelial activation in ischaemic CVD patients despite treatment with antithrombotic therapy.

Comparison of von Willebrand factor antigen, von Willebrand factor‐cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura

Summary.  Replacement of normal levels of von Willebrand factor‐cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD‐ and MB‐treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.

Assessment of the antiplatelet effects of low to medium dose aspirin in the early and late phases after ischaemic stroke and TIA

Vascular events commonly recur in stroke patients on aspirin, and may reflect incomplete inhibition of platelet function with aspirin therapy. The platelet function analyser (PFA-100) activates platelets by aspirating a blood sample at a moderately high shear rate through a capillary to a biologically active membrane with a central aperture. The membrane is coated with collagen, and either ADP (C-ADP) or epinephrine (C-EPI). The time taken for activated platelets to adhere, aggregate, and occlude the aperture is called the closure time. Previous studies have shown that aspirin prolongs the C-EPI closure time, without prolongation of the C-ADP closure time, in the majority of control subjects. We hypothesised that the PFA-100 would provide a sensitive assay for the detection of early and convalescent phase cerebrovascular disease (CVD) patients who had incomplete inhibition of platelet function with aspirin. We investigated potential cyclooxygenase-dependent and -independent mechanisms that might influence the responsiveness to aspirin using the PFA-100. Patients were studied during the early (≤4 weeks, n = 57) and convalescent phases (≥3 months, n = 46) after ischaemic stroke or TIA. To investigate potential mechanisms that could contribute to aspirin responsiveness on the PFA-100, we measured von Willebrand factor antigen levels, and carried out platelet aggregometry experiments in platelet-rich plasma in response to sodium arachidonate (1 mM) and ADP (5 µM). Sixty percent of patients in the early phase and 43% of patients in the convalescent phase did not have prolonged C-EPI closure times on 75–300 mg of aspirin daily, and were defined as aspirin non-responders. Median C-ADP closure times were significantly shorter in aspirin non-responders than aspirin-responders in both the early and convalescent phases after symptom onset (P ≤ 0.008), suggesting platelet hyper-reactivity to collagen or ADP in the aspirin non-responder subgroup. There was a significant inverse relationship between plasma von Willebrand factor antigen levels and C-EPI closure times in both early and convalescent phase CVD patients (P ≤ 0.008). Mean von Willebrand factor antigen levels were significantly higher in aspirin non-responders than aspirin responsive patients in the early (P = 0.001), but not convalescent phase (P = 0.2) after stroke and TIA. None of the patients studied were defined as being aspirin-resistant using sodium arachidonate- or ADP-induced platelet aggregometry. A large proportion of ischaemic CVD patients have incomplete inhibition of platelet function with low to medium dose aspirin using the PFA-100. The results suggest that cyclooxygenase-independent mechanisms, including elevated von Willebrand factor antigen levels, play an important role in mediating aspirin non-responsiveness on the PFA-100.

Increased platelet count and leucocyte–platelet complex formation in acute symptomatic compared with asymptomatic severe carotid stenosis

Objective: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. Methods: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte–platelet complexes in patients with acute (0–21 days, n = 19) and convalescent (79–365 days) symptomatic (n = 16) and asymptomatic (n = 16) severe (⩾70%) carotid stenosis. Most patients were treated with aspirin (37.5–300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. Results: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil–platelet (p = 0.004), monocyte–platelet (p = 0.046), and lymphocyte–platelet complexes (p = 0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil–platelet and monocyte–platelet complexes (p = 0.03) were higher in acute symptomatic (n = 11) than asymptomatic patients (n = 14). In the convalescent phase, the median percentages of all leucocyte–platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. Conclusion: Increased platelet count and leucocyte–platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.

The clinical utility of ADAMTS13 activity, antigen and autoantibody assays in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) has been linked to a severe deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. Since the identification of ADAMTS13, and its target cleavage sequence in von Willebrand factor (VWF), several novel ADAMTS13 activity, antigen and autoantibody assays have been developed. Our aim was to evaluate the potential use of these novel assays. ADAMTS13 activity and inhibitors were measured by overnight incubation of patient plasma with pure VWF followed by multimer or collagen binding analysis. ADAMTS13 activity (Rapid peptide assay), antigen and immunoglobulin G anti‐ADAMTS13 were measured by enzyme‐linked immunosorbent assay. 118 samples from seven TTP patients (six adult idiopathic, one congenital) were studied longitudinally during episodes of TTP, their treatment and prophylaxis. ADAMTS13 antigen levels varied considerably between patients and sample times, but in new cases of acute TTP, rapid assays of ADAMTS13 antigen, on serial samples, maybe helpful in confirming the diagnosis. The rapid peptide ADAMTS13 activity assay showed good concordance of results with the older activity assay methods. The change in ADAMTS13 activity mirrored the autoantibody level and in 5/6 acquired TTP cases, a fall in antibody appeared to predict a rise in ADAMTS13 activity, potentially allowing modification of patient management based on autoantibody levels.

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique

Summary. This study assessed the inter‐laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio ∼2·0 and ∼4·0] and a further two subjects receiving treatment with unfractionated heparin (anti‐Xa 0·07 iu/ml and 0·31 iu/ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra‐assay CV < 9·5%, inter‐assay CV < 12·5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom® TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0·96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL‐300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.