Andrew S. Lawrie

Microparticle sizing by dynamic light scattering in fresh-frozen plasma

Background   We have previously shown that fresh‐frozen plasma (FFP) contains red blood cell‐derived procoagulant microparticles (MPs) that are removable by 0·2 µm filtration. Given the limitations of current methods for accurately sizing MPs, we have applied the novel approach of dynamic light scattering (DLS) to characterize the size distributions of these MPs within FFP.

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.

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.

The quality of fresh-frozen plasma produced from whole blood stored at 4°C overnight

BACKGROUND: The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4°C overnight is adequate for its intended purpose.

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.

Coagulation factor levels in cryosupernatant prepared from plasma treated with amotosalen hydrochloride (S-59) and ultraviolet A light

BACKGROUND: The standard treatment for thrombotic thrombocytopenic purpura (TTP) is plasma exchange with fresh‐frozen plasma (FFP). Exposure to large volumes of FFP increases the risk of transfusion‐transmitted infections. Cryosupernatant (CSP) offers a theoretical advantage over FFP, because it lacks the large von Willebrand factor (VWF) forms implicated in the pathogenesis of TTP. This study compared the hemostatic variables of CSP prepared from FFP treated with a photochemical pathogen inactivation process to CSP prepared from conventional FFP.

The dynamics of clot formation in fresh-frozen plasma.

Background  Factor VIII (FVIII) levels are used as a quality marker of fresh‐frozen plasma (FFP); however, other clotting factors are not routinely measured.

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 impact of elective knee/hip replacement surgery and thromboprophylaxis with rivaroxaban or dalteparin on thrombin generation

Total hip/knee replacement surgeries are associated with an increased risk of venous thromboembolism and post‐operative thromboprophylaxis has become standard treatment. This study aimed to: (i) assess the impact of hip/knee replacement surgery on ex vivo thrombin generation (TG), prothrombin fragments 1 + 2 (F1 + 2), thrombin‐antithrombin complexes (TAT) and D‐dimer; (ii) compare the anticoagulant effects of dalteparin and rivaroxaban on TG 24 h after surgery. Haemostatic variables were assessed in plasma samples of 51 patients taken pre‐operatively, peri‐operatively, and 24 h post‐operatively. Prophylaxis, once a day, with dalteparin or rivaroxaban, starting 6–8 h post‐operatively, was administered in 25 (14 knee/11 hip) and 26 patients (13 knee/13 hip) respectively. TG, F1 + 2, TAT and D‐dimer increased during surgery. Dalteparin patients showed a variable TG response 24 h after surgery: conversely, the effect of rivaroxaban on TG was consistent across individuals. Good correlation was seen between rivaroxaban levels and TG‐lag‐time (rs = 0·46, P = 0·01); TG‐time‐to‐Peak (rs = 0·53, P = 0·005); TG‐peak‐thrombin (rs = −0·59, P = 0·001); and TG‐velocity‐index‐rate (rs = −0·61, P = 0·0009). Patients who received rivaroxaban showed a greater decrease of TG, F1 + 2 and TAT (but not D‐dimer) than those on dalteparin. TG increases during hip/knee replacement surgery. Rivaroxaban inhibits TG more than dalteparin at 24 h after surgery.

Porcine platelets contain an increased quantity of ultra‐high molecular weight von Willebrand factor and numerous alpha‐granular tubular structures

Summary. Immunoelectronmicroscopy of human platelet α‐granules reveals that von Willebrand factor (vWf:Ag) colocalizes with a small number of discrete tubular structures which appear identical to those observed within the WeibelPalade bodies of endothelial cells. Although it is likely that tubules are composed of vWf:Ag as they are absent in severe vWD porcine platelets, their exact structural and functional nature is still unclear. In this study quantitative/qualitative analysis of vWf:Ag was undertaken in a series of platelet preparations obtained from normal pigs, normal humans and various vWD patients. Electron microscopy confirmed that normal pig platelet α‐granules contain numerous, regularly spaced tubular structures eccentrically located and coincident with immunogold staining of vWf:Ag. In contrast, normal human platelet α‐granules contain significantly fewer tubules (usually four to six) which are absent or reduced in number within various vWD platelet sections. Furthermore, the pig platelet lysates not only contained a full complement of multimers but also demonstrated significant intense staining of ultra‐high MW material, irrespective of the presence or absence of proteolytic inhibitors. This ultrahigh MW vWf appears similar to that observed within lysates prepared from endothelial cells and is susceptible to degradation to lower MW multimers. This study suggests that the tubular structures within α‐granules and Weibel‐Palade bodies may be composed of, or structurally related to, the ultra‐high MW intracellular form of vWf:Ag.