Lorenzo Alberio

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional α-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as ‘COAT-platelets’, bind additional α-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and α2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

Thrombotic thrombocytopenic purpura

Summary.  This overview summarizes the history of thrombotic thrombocytopenic purpura (TTP) from its initial recognition in 1924 as a most often fatal disease to the discovery in 1997 of ADAMTS‐13 deficiency as a major risk factor for acute disease manifestation. The cloning of the metalloprotease, ADAMTS‐13, an essential regulator of the extremely adhesive unusually large von Willebrand factor (VWF) multimers secreted by endothelial cells, as well as ADAMTS‐13 structure and function are reviewed. The complex, initially devised assays for ADAMTS‐13 activity and the possible limitations of static in vitro assays are described. A new, simple assay using a recombinant 73‐amino acid VWF peptide as substrate will hopefully be useful. Hereditary TTP caused by homozygous or double heterozygous ADAMTS‐13 mutations and the nature of the mutations so far identified are discussed. Recognition of this condition by clinicians is of utmost importance, because it can be easily treated and – if untreated – frequently results in death. Acquired TTP is often but not always associated with severe, autoantibody‐mediated ADAMTS‐13 deficiency. The pathogenesis of cases without severe deficiency of the VWF‐cleaving protease remains unknown, affected patients cannot be distinguished clinically from those with severely decreased ADAMTS‐13 activity. Survivors of acute TTP, especially those with autoantibody‐induced ADAMTS‐13 deficiency, are at a high risk for relapse, as are patients with hereditary TTP. Patients with thrombotic microangiopathies (TMA) associated with hematopoietic stem cell transplantation, neo‐plasia and several drugs, usually have normal or only moderately reduced ADAMTS‐13 activity, with the exception of ticlopidine‐induced TMA. Diarrhea‐positive‐hemolytic uremic syndrome (D+ HUS), mainly occurring in children is due to enterohemorrhagic Escherichia coli infection, and cases with atypical, D− HUS may be associated with factor H abnormalities. Treatment of acquired idiopathic TTP involves plasma exchange with fresh frozen plasma (FFP), and probably immunosuppression with corticosteroids is indicated. We believe that, at present, patients without severe acquired ADAMTS‐13 deficiency should be treated with plasma exchange as well, until better strategies become available. Constitutional TTP can be treated by simple FFP infusion that rapidly reverses acute disease and – given prophylactically every 2–3 weeks – prevents relapses. There remains a large research agenda to improve diagnosis of TMA, gain further insight into the pathophysiology of the various TMA and to improve and possibly tailor the management of affected patients.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories.

INTRODUCTION Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Protein C Replacement in Severe Meningococcemia: Rationale and Clinical Experience

Severe meningococcemia, which is associated with hemodynamic instability, purpura fulminans and disseminated intravascular coagulation, still has a high mortality rate, and patients who survive are often left invalids because of amputations and organ failure. Clinical studies have shown that levels of protein C are markedly decreased in patients with severe meningococcemia and that the extent of the decrease correlates with a negative clinical outcome. There is a growing body of data demonstrating that activated protein C, in addition to being an anticoagulant, is also a physiologically relevant modulator of the inflammatory response. The dual function of protein C may be relevant to the treatment of individuals with severe meningococcal sepsis. In the present review we give a basic overview of the protein C pathway and its anticoagulant activity, and we summarize experimental data showing that activated protein C replacement therapy clearly reduces the mortality rate for fulminant meningococcemia.

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes

BackgroundA concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency.MethodsIn 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers.ResultsPatients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate.ConclusionThe positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.Trial registrationClinicalTrials.gov NCT00718289

Titre of anti-heparin/PF4-antibodies and extent of in vivo activation of the coagulation and fibrinolytic systems

Heparin-induced thrombocytopenia (HIT) is mediated by antibodies directed against the heparin/platelet factor 4 (PF4) complex. Our aim was to investigate whether the antibody titre is associated with the degree of in vivo thrombin generation. We measured the anti-heparin/PF4-antibody titre, prothrombin fragments F1+2, thrombin-antithrombin (TAT) complexes and D-dimers in plasma samples from 225 patients with suspected HIT. Antibody titres as detected by a particle gel immunoassay strongly correlated with optical density values measured by ELISA (r=0.84, p<0.0001). Patients with titres > or =4 (n=44) had significantly higher median levels of F1+2 (2.49 nmol/l), TAT (13.01 microg/l) and D-dimers (3340 microg/l) compared to patients with undetectable antibodies (n=148; F1+2 1.61 nmol/l, TAT 4.95 micro g/l, D-dimers 1911 micro g/l; p<0.0001 for all comparisons) or patients with titres of 1-2 (n=33; F1+2 1.44 nmol/l, p=0.0014; TAT 4.37 microg/l, p=0.0018; D-dimers 2231 microg/l, p=0.0016). Multivariate analysis indicated the anti-heparin/PF4-antibody titre as an independent predictor for F1+2 (p=0.0036), TAT (p=0.0176) and D-dimer (p=0.0003) levels. This relationship remained statistically significant after exclusion of patients with concomitant prothrombotic conditions and/or thromboembolic complications during heparin treatment. These data demonstrate that high anti-heparin/PF4-antibody titres are independently associated with an increased in vivo thrombin generation. Rapid determination of the anti-heparin/PF4-antibody titre could help guide clinical management, identifying a subset of HIT-patients who are at high risk of developing thromboembolic complications and possibly require alternative anticoagulation in therapeutic dosage even in the context of isolated HIT.

Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature.

Many preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4-6, 8-12, 24-28 and 48-52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C, VWF:RCo, VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24-28 hours, and for PT, aPTT and FVII:C at 48-52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance). The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factor V and VIII coagulant activity, and total PS antigen all investigated parameters can be measured 24-28 hours after specimen collection without observing clinically relevant changes.

D-Dimers Predict Stroke Subtype when Assessed Early

Background:Early classification of ischemic stroke subtype is important for secondary stroke prevention and may guide further investigations. Methods: Levels of coagulation activation [fibrinopeptide A (FPA), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT)] and fibrinolysis activation [plasmin-α2-antiplasmin complex (PAP), D-dimers] markers were measured in 98 consecutive patients with a first-ever acute ischemic stroke admitted within 12 h after symptom onset. Results: Median age was 67 years and 44% were women. Median time from symptom onset to blood sampling was 4 h. Stroke subtype was classified as ‘cardioembolic’ (54%), ‘large-artery atherosclerosis’ (11%), ‘small-vessel disease’ (5%), ‘other determined’ (9%) or ‘undetermined etiology’ (20%). Patients with cardioembolic stroke suffered more often from coronary artery disease than patients with other stroke etiologies (40 vs. 22%, p = 0.019). There were no differences in age, sex, stroke severity, time to blood sampling, frequency of hypertension, diabetes mellitus or current smoking. D-dimers (medians) were higher in patients with cardioembolic strokes than in those with other etiologies (615 vs. 322 μg/l, p < 0.001). No differences in F1+2, FPA, TAT or PAP levels were found. After multivariate analysis, higher D-dimer levels remained independently associated with cardioembolic stroke (p = 0.022). When measured within 6 h, D-dimers below 300 μg/l excluded cardioembolic stroke with a sensitivity of 100% and a specificity of 52%. Conclusions: Low D-dimer levels in the first few hours make a cardioembolic stroke unlikely, and may be useful to guide further investigations. Other coagulation markers were not useful in differentiating between different stroke etiologies.

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real‐time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440‐13_c.1440‐1del in intron 14 of ITGA2B causing exon skipping in seven unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits, thereby interfering with integrin maturation and/or function. Our study extends knowledge of GT and the pathophysiology of an integrin.

Dosing lepirudin in patients with heparin-induced thrombocytopenia and normal or impaired renal function: a single-center experience with 68 patients

The recommended dose (bolus 0.4 mg/kg followed by 0.15 mg/kg per hour) of lepirudin, a direct thrombin inhibitor licensed for treatment of heparin-induced thrombocytopenia (HIT), is too high. Starting in 2001, we omitted the bolus and reduced maintenance dose by at least one-third. Analyzing 53 HIT patients treated between January 2001 and February 2007, we observed that therapeutic anticoagulation intensity already 4 hours after lepirudin start had been reached with the following initial lepirudin doses (median): 0.078 mg/kg per hour [creatinine clearance (CrCl) more than 60 mL/min], 0.040 mg/kg per hour (CrCl 30-60 mL/min), and 0.013 mg/kg per hour (CrCl < 30 mL/min). The efficacy of this treatment was documented by increasing platelets and decreasing D-dimers. Based on this experience, we derived a lepirudin dosing regimen, which was prospectively evaluated treating 15 HIT patients between March 2007 and February 2008. We show that omitting the initial lepirudin bolus and administering 0.08 mg/kg per hour in patients with CrCl more than 60 mL/min, 0.04 mg/kg per hour in patients with CrCl 30-60 mL/min, and 0.01 to 0.02 mg/kg per hour in those with CrCl less than 30 mL/min is efficacious and safe, as documented by increasing platelet counts, decreasing D-dimer levels, and rare thrombotic (1 of 46) and major bleeding (4 of 46) complications.