Ingrid Skornova

Progress in the Understanding of Sticky Platelet Syndrome.

&NA; The knowledge on the etiology of thrombosis has increased tremendously over the past decades. Nevertheless, Virchow triad is still traditionally invoked to explain mechanisms leading to thrombosis, alleging concerted roles for abnormalities in blood composition, vessel wall components, and blood flow in the development of arterial and venous thrombosis. Recent decades have been focused primarily on describing abnormalities in blood composition, including defects of coagulation proteins and platelets. Although defects of coagulation factors are relatively well‐described in the literature, prothrombotic platelet disorders are still less understood. One such defect, the Wien‐Penzing defect was first described in 1991. Another platelet defect is sticky platelet syndrome (SPS). In this article, we review information about SPS, and we propose a new definition and standardization of diagnostic criteria. We also attempt to explain the causes and consequences of this condition.

Identification of Two Novel Fibrinogen Bβ Chain Mutations in Two Slovak Families with Quantitative Fibrinogen Disorders

Congenital fibrinogen disorders are caused by mutations in one of the three fibrinogen genes that affect the synthesis, assembly, intracellular processing, stability or secretion of fibrinogen. Functional studies of mutant Bβ-chains revealed the importance of individual residues as well as three-dimensional structures for fibrinogen assembly and secretion. This study describes two novel homozygous fibrinogen Bβ chain mutations in two Slovak families with afibrinogenemia and hypofibrinogenemia. Peripheral blood samples were collected from all subjects with the aim of identifying the causative mutation. Coagulation-related tests and rotational thromboelastometry were performed. All exons and exon–intron boundaries of the fibrinogen genes (FGA, FGB and FGG) were amplified by PCR followed by direct sequencing. Sequence analysis of the three fibrinogen genes allowed us to identify two novel homozygous mutations in the FGB gene. A novel Bβ chain truncation (BβGln180Stop) was detected in a 28-year-old afibrinogenemic man with bleeding episodes including repeated haemorrhaging into muscles, joints, and soft tissues, and mucocutaneous bleeding and a novel Bβ missense mutation (BβTyr368His) was found in a 62-year-old hypofibrinogenemic man with recurrent deep and superficial venous thromboses of the lower extremities. The novel missense mutation was confirmed by molecular modelling. Both studying the molecular anomalies and the modelling of fibrinogenic mutants help us to understand the extremely complex machinery of fibrinogen biosynthesis and finally better assess its correlation with the patient’s clinical course.

Role of VASP Phosphorylation Assay in Monitoring the Antiplatelet Therapy

Abstract Dual antiplatelet treatment with clopidogrel and aspirin represents standard regimen in prevention of thromboembolic events in patients with ischemic heart disease undergoing percutaneous coronary intervention (PCI). One of the greatest pitfalls of clopidogrel treatment is large inter-individual variability in response. Large amount of patients does not respond adequately and therefore are not „protected“ even in spite of receiving the therapy. Poor responders are exposed to three-fold increased risk of myocardial infarction, stent thrombosis and cardiac death. Clopidogrel is an antiplatelet prodrug, whose active metabolite inhibits platelet function by irreversible binding to the (adenosine diphosphate) platelet receptor P2Y12. Receptor P2Y12 plays primal role in ADP-mediated platelet activation, and also in mechanism of action of ADP inhibitors such as clopidogrel, prasugrel etc. Reasons stated above, raised the necessity for implementing reliable laboratory test in order to identify the unprotected patients. In an ideal scenario, such test would serve to adjust the dose and guide the individual tailored treatment. Vasodilator Stimulated Phosphoprotein (VASP) is an intracellular platelet protein which is non phosphorylated at basal state. Since its relation in cascade with P2Y12 receptor, VASP phosphorylation corerlates with inhibition of P2Y12 which is the receptor of prime importance in ADP mediated activation of platelets and as is primary target of ADP inhibitors action. Outcome of the assay is represented as the value of platelet reactivity index (PRI), where PRI values above 50% are considered inadequate response to treatment and signal exposure to increased risk of myocardial infarction, post-PCI stent thrombosis and cardiac death. VASP-P flow cytometric assay is emerging into the spotlight as the promising method, mostly for its specificity for ADP inhibitors, better outlook for standardising results and lesser sample manipulation compared to multiple electrode aggregometry.

Measurement of platelet p-selectin expression by flow cytometry in patients with acute ischemic stroke

Abstract Aims: The aim of this study was to asses the platelet activation in the acute phase of ischemic stroke and transient ischemic attack (TIA) by defining p-selectin (CD62) expression by flow cytometry in vivo – without stimulation with agonists. We also studied whether antiplatelet therapy supresses the levels of baseline p-selectin expression and verified if there is a correlation between platelet CD62 expression and the type of ischemic stroke. Methods: We determined the expression of platelet surface p-selectin using whole-blood flow cytometry within the first 48-hours after onset of cerebral symptoms in patients with atherothrombotic and lacunar ischemic stroke and in healthy volunteers. We studied the realationship between antiplatelet medication and the type of ischemic stroke to baseline p-selectin expression. Results: Patients with acute cerebral ischemia have an excess of circulating platelets that express p-selectin, compared to healthy volunteers. The difference between average p-selectin expression in the group of healthy volunteers and the group of patients with stroke was statistically significant (p-value < 0,000001). Patients with stroke without antiplatelet medication showed a higher p-selectin expression than patients with antiplatelet medication (ASA, CLP, or ASA and CLP), hovewer, the difference was not statistically significant. There is no relationship between CD62 expression and the type of stroke. Conclusions: We can conclude that p-selectin is a highly sensitive blood biomarker of increased platelet activation. Antiplatelet therapy suppresses baseline p-selectin expression only minimally, insignificantly according to our results.

Successful Use of a Highly Purified Plasma von Willebrand Factor Concentrate Containing Little FVIII for the Long-Term Prophylaxis of Severe (Type 3) von Willebrand's Disease

We read with great interest the article by Curnow et al,1 describing treatment of von Willebrand disease (VWD), as published in a recent issue of the journal, together with other interesting articles related to such disorders, as appearing in a recent issue of Seminars in Thrombosis & Hemostasis. We thereforewish to report our experience of thefirst successful use of a highly purified plasma von Willebrand factor (VWF) concentrate containing little FVIII in the long-term prophylaxis of severe vWD in Slovakia. VWD is the most common inherited bleeding disorder caused by a quantitative and/or qualitative abnormality in the adhesive plasma protein VWF.2 VWF represents a highmolecular-weight adhesive glycoprotein that plays an essential role in the primary hemostasis by promoting platelet adhesion to the subendothelium and platelet plug formation at the sites of vascular injury. VWD is classified into three major types: partial quantitative deficiency (type 1), qualitative deficiency (type 2), and complete quantitative deficiency (type 3). VWD type 2 is further divided into four variants (2A, 2B, 2N, 2M), based on the characteristics of the dysfunctional VWF.1,3 Individuals presenting with clinical manifestations of VWDmay have amild,moderate, or severe bleeding tendency since childhood, usually proportional to thedegree of theVWF deficiency/defect.4 There are two principle treatments of choice in VWD: desmopressin (DDAVP) and substitution therapywith bloodproducts containing VWF (with/without factor VIII; FVIII). Other forms of treatment can be considered as adjunctive or alternative to those mentioned above.5 We present amale patient (now aged 34 years) with VWD type 3 (►Table 1). Our study was conducted in accordance with the Declaration of Helsinki. It was also approved by the institutional ethics committee on human research and informed consent was obtained. Early after birth, several spontaneous bleeding manifestations presented, which, over subsequent years, continued with many mucosal and joint bleeding episodes; these promoted the development of arthropathy. Substitution therapy was historically administered “on demand” during these events. From 1999, the then 17-year-old patient was subsequently treated with regular prophylactic administration of VWF/FVIII plasma-derived concentrates. Despite preventive administration, the patient continued to have recurrent mucosal bleeding and epistaxis, albeit with less frequent bleeding into joints. FromDecember 2015, he was switched from a concentrate containing both VWF and FVIII to a highly purified plasma VWF concentrate containing little FVIII (Willfact; LFB Biomedicaments, France). Levels of plasma VWF and FVIII in individuals with VWD can be regulated by the infusion of plasma-derived concentrates containing either VWF alone or VWF with FVIII.1 Available concentrates contain different amounts of VWF and FVIII, and the appropriate dosage should, therefore, be defined according to the specific product’s characteristics.6 The primary symptoms of the severe form of VWD, comprising deficiency of both VWFand FVIII, may therefore comprise frequent hemophilia-like joint bleeds or recurrent spontaneous mucosal bleeding (e.g., epistaxis and gastrointestinal bleeding). Therefore, in patients with frequent bleeding, the treatment rationale is use of secondary long-term prophylaxis.6,7 Although patients with severe VWD produce FVIII, Letter to the Editor

Review of the Pharmacology of the Emerging Possibilities of the Direct Oral Anticoagulants' Reversal

BACKGROUND Direct oral anticoagulants (DOACs) offer consistent and predictable anticoagulation, oral administration with good patient compliance and a good safety profile. Dabigatran - a direct thrombin inhibitor, apixaban and rivaroxaban - direct factor Xa inhibitors are now largely used for anticoagulation in patients with nonvalvular atrial fibrillation and in patients with venous thromboembolism. These agents have emerged as an expediential clinical choice in long-term anticoagulation for an increasing number of patients. Despite their advantages, concerns persist about a lack of rapid reversal agents in urgent clinical situations. METHODS This review is focused on the pharmacology of nonspecific and target-specific reversal agents for DOACs-induced anticoagulation. A systemic review of preclinical and clinical studies published in peer-reviewed scientific journals was performed. RESULTS AND CONCLUSIONS Fresh frozen plasma and coagulation factors concentrates might be considered in bleeding emergencies; however, there is a lack of larger studies confirming the efficacy of coagulation factors concentrates for the reversal of DOACs-induced anticoagulation, and a particular risk of coagulation factors concentrates-induced thrombosis. Recently, idarucizumab has been approved commercially for acute reversal of dabigatran in emergencies as a first target-specific reversal agent. Moreover, andexanet alpha and aripazine are being extensively studied in several phase II and III clinical studies. It is likely that more target-specific agents for reversal of DOACs-induced anticoagulation will be introduced to clinical practice in near future.

Monitoring of Hemostasis and Management of Anticoagulant Thromboprophylaxis in Pregnant Women with Increased Risk of Fetal Loss.

Physiological prothrombotic changes during pregnancy and the postpartum period, along with other preexisting maternal risk factors, increase the risk of both venous thromboembolism (VTE) and adverse pregnancy outcomes. Pregnancy complications that develop due to placental insufficiency as a result of inappropriate activation of coagulation are present in more than 5% of pregnancies and can contribute to significant maternal morbidity and mortality. Therefore, anticoagulant prophylaxis in women with congenital and acquired thrombophilic conditions should be actively considered. According to the Guidelines of American College of Chest Physicians, the use of low-molecular-weight heparin is suggested for prophylaxis of VTE and pregnancy complications in high-risk pregnant women. However, personalized refinements of such thromboprophylaxis remains unspecified, despite the necessity of better targeted recommendations for life-threatening conditions. We, therefore, review the possibilities of longitudinal monitoring and comprehensive assessment of changes in hemostasis in the group of high-risk pregnant women, which can then be used for early prediction and individualization of the optimal anticoagulant thromboprophylaxis of pregnancy complications. Simultaneously, we present our single-center experience with such monitoring and our first series of results.

Thrombelastography and Thrombelastometry

Abstract The term thrombelastography / thrombelastometry was used to describe the trace produced from measurement of the viscoelastic changes associated with fibrin polymerization. The result of measurement is a compact mapping of the various stages of haemostasis. One of the first real clinical applications of this method was the haemostatic monitoring of liver transplantation and cardiac surgery using extracorporeal circulation. In trauma patients the thrombelastography /thrombelastometry was proved to predict early transfusion requirements. Another authors suggest thrombelastography /thrombelastometry as a possible tool for early identification of pregnant women at increased risk of fetal loss. This article provides overview on the development of thrombelastography / trombelastometry and its possible use in laboratory of haemostasis.