Jasper A. Remijn

Platelet Activation Test in Unprocessed Blood (Pac-t-UB) to Monitor Platelet Concentrates and Whole Blood of Thrombocytopenic Patients.

Background: Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients. Methods: We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient. Results: Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage. Discussion: Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region*

Summary. The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12‐carboxy‐terminal residues of the γ‐chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the γ‐chain segments γ318–320 and γ408–411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to γΔ408‐411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: γΔ319‐320, γ318Asp→Ala and γ320Asp→Ala. Furthermore, antibodies raised against the sequences γ308–322 and γ316–333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment γ316–322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin‐specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

Strenuous exercise induces a hyperreactive rebalanced haemostatic state that is more pronounced in men

Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.

Diagnostic choices and clinical outcomes in octogenarians and nonagenarians with iron-deficiency anemia in the Netherlands.

To evaluate current clinical practice for octogenarians with iron‐deficiency anemia (IDA) by assessing referral patterns, diagnostic choices, clinical consequences of omission of endoscopy, and risks and benefits of IDA‐related surgery.

Citalopram is a more potent platelet function inhibitor than paroxetine in a case–control study

Serotonin is a major component of platelet dense granules [1]. It mediates vasoconstriction and supports platelet activation via platelet 5-hydroxytryptamine receptors [2,3]. Platelets take up serotonin via serotonin transporters in their membranes. This uptake is inhibited by selective serotonin reuptake inhibitors (SSRIs) [4], which thereby affect platelet function [4–6]. Some studies have shown that SSRIs reduce the risk of cardiovascular disease events [7–10], although there is still some controversy on the subject [11,12]. In line with this, numerous studies have shown an increased bleeding risk with SSRI use, although not all studies could confirm this (for an overview of the literature, see [13]). Studies on the influence of SSRIs on platelet function have been performed mainly ex vivo [14–16] or in patient groups treated with with different SSRIs, but analyzed together [5,6,17]; different types of SSRI have been reported to differ in therapeutic efficacy [18]. We compared the effects of paroxetine and citalopram on platelet responsiveness in a case–control study of 13 patients with a platelet function analyzer, and a platelet responsiveness assay to investigate the hypothesis that these SSRIs have different effects on platelet function. Thirteen patients and 20 controls were enrolled in the present study from September to January 2011. After informed consent had been obtained, venous blood was collected from patients and healthy volunteers in vacuum tubes with 3.2% trisodium citrate (Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Patients were included at GGNet Mental Health in Apeldoorn, The Netherlands. The control group was slightly younger and contained more women than the patient group (Table 1). Patients were taking either paroxetine or citalopram on a therapeutic basis, but no other antidepressants or any antiplatelet medication (aspirin or clopidogrel). Drug levels (citalopram, desmethylcitalopram, and paroxetine) were measured with ultra-performance liquid chromatography (ACQUITY UPLC) and tandem mass spectrometry (ACQUITY TQD; Waters Chromatography, Etten-Leur, The Netherlands); on average, drug levels were within the therapeutic range. Platelet P-selectin expression in whole blood was determined in response to serial dilutions of ADP ranging from 0.008 to 125 lM, cross-linked collagen-related peptide (CRPXL) ranging from 0.0002 to 2.5 lg/mL, the thromboxane analog U-46619 ranging from 0.8 to 12500 lg/mL, and the thrombin receptor agonist SFLLRN (TRAP) ranging from 0.038 to 625 lM (see also [19]). All samples were analyzed on a FACSCalibur flow cytometer (Beckton Dickinson, Breda, the Netherlands) on the day of processing. Single platelets were gated on the basis of forward-scatter and side-scatter properties, and their median fluorescence intensity was measured. Dose–response graphs and areas under the curves expressed in arbitrary units were produced with PRISM 5.01 software (Graphpad Software, La Jolla, CA, USA). Nonparametric tests were used to test the difference between the different groups, with SPSS 18.0 (PASW Statistics, Hong Kong, China). Platelet P-selectin expression was significantly reduced in patients using citalopram for the P2Y12 receptor agonist ADP, the glycoprotein VI receptor agonist CRP-XL, and the protease-activated receptor-1 agonist TRAP, whereas paroxetine use did not affect platelet reactivity relative to the control group (Table 1). There were no significant differences between the groups in the area under the curve of the thromboxane receptor agonist U-46619. The platelet function analyzer tests (PFA-100; Siemens Diagnostics, Breda, The Netherlands), performed according to the manufacturer s directions, did not show any differences between any of the groups (Table 1). For whole blood serotonin measurements, citrated blood stored at ) 80 C was thawed, and perchloric acid was added up to 0.6 M; the mixture was then vortexed and centrifuged at Correspondence: Thijs C. van Holten, Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands. Tel.: +31 88 7557008; fax: +31 88 7555418. E-mail: tholten@umcutrecht.nl

Molecular basis of congenital afibrinogenaemia in a Dutch family

&NA; Congenital afibrinogenaemia is a rare autosomal recessive disorder characterized by complete absence or trace amounts of fibrinogen. Here we report the identification of the molecular defect underlying afibrinogenaemia in a Dutch patient. DNA sequence analysis of the fibrinogen A&agr;, B&bgr; and &ggr;‐genes revealed a homozygous deletion of two adenines between nucleotides 3120 and 3122 in exon 4 of the gene coding for the A&agr;‐chain. This deletion results in a frameshift with a predicted premature end of translation at codon 140. This is the first report of a patient homozygous for this rare mutation associated with afibrinogenaemia. Blood Coagul Fibrinolysis 14:299‐302 & 2003 Lippincott Williams & Wilkins.

Impaired platelet adhesion to lysed fibrin, whereas neutrophil adhesion remains intact under conditions of flow

Vessel wall injury induces the formation of a haemostatic plug. Restoration of vascular integrity should involve cessation of further platelet and fibrin deposition and subsequent removal of these thrombi by both the fibrinolytic system and proteases delivered by infiltrating inflammatory cells. We hypothesized that adhesion of platelets and inflammatory cells [polymorphonuclear leucocyte (PMN)] to fibrin is differently supported after exposure of fibrin during fibrinolysis. Fibrin surfaces were exposed to fibrinolytic agents, and platelet and PMN adhesion was studied under conditions of flow. Specific adhesion of platelets to preformed fibrin was reduced by fibrinolytic treatment of the fibrin. PMN adhesion to fibrin was only slightly affected even after 180 min exposure to plasmin. With fibrin still present after fibrinolytic treatment, the impaired platelet adhesion seems explained by loss of the primary platelet adhesion site γ400–411 on fibrin. PMN binding to fibrin clearly depends on other sites that are less degraded by fibrinolysis. We have shown that PMN adhesion in flowing blood to lysed fibrin was still present, whereas platelet adhesion was impaired due to the loss of the primary platelet adhesion site γ400–411. Based on our in-vitro perfusion model, we conclude that fibrinolysis specifically interferes with the thrombogenicity of fibrin in the haemostatic plug, whereas the inflammatory response is preserved. The latter may participate in the long-term removal and restructuration of the plug.

Hypobaric Hypoxia Causes Elevated Thrombin Generation Mediated by FVIII that is Balanced by Decreased Platelet Activation

INTRODUCTION Epidemiological studies suggest that hypobaric hypoxia at high altitude poses a risk for developing venous thromboembolism. The cause of this observed hypercoagulability remains unclear. Therefore, this study aimed to investigate the effect of hypobaric hypoxia at 3,883 m above sea level on thrombin generation and platelet activation. METHODS After complying with medical ethical procedures, 18 participants were recruited, of whom 1 had to leave the study prematurely due to mild acute mountain sickness. Blood was drawn first at 50 m above sea level and second at 3,883 m altitude after gradual acclimatization for 6 days. Thrombin generation was measured in whole blood, platelet-rich plasma and platelet-poor plasma. Platelet activation was assessed using a whole blood flow-cytometric assay. Coagulation factor levels, D-dimer levels and markers of dehydration and inflammation were measured. RESULTS Hypobaric hypoxia at 3,883 m altitude caused increased thrombin generation, measured as peak height and endogenous thrombin potential, in whole blood, platelet-rich and platelet-poor plasma without or at low tissue factor concentration. The elevated thrombin generation was mediated by increased factor VIII levels and not caused by dehydration or inflammation. In contrast, spontaneous and agonist-induced platelet activation was decreased at high altitude. CONCLUSION Hypobaric hypoxia causes increased factor VIII-mediated thrombin generation. The hypercoagulability was balanced by decreased platelet activation. These findings may explain why venous, and not arterial thrombotic events occur more frequently at high altitude.

High prevalence of reduced thrombin generation and/or decreased platelet response in women with unexplained heavy menstrual bleeding

Heavy menstrual bleeding (HMB) is a condition that affects 20%‐30% of women of reproductive age. HMB has a multifactorial pathophysiology, which is incompletely understood. HMB symptoms are very common in patients with established haemostasis defects, likewise, women with heavy menstrual bleeding have a higher prevalence of impaired Von Willebrand factor (VWF) levels and function, thrombocytopenia, impaired platelet function and impaired coagulation. The aim of this study was to quantify the prevalence of impaired platelet function, impaired coagulation and reduced VWF activity in patients with HMB.