Diagnostics of thrombocytopenias

Laboratory methods used for the diagnostics of thrombocytopenias are reviewed. Differential diagnosis is usually carried out between immune and hypoproductive forms of thrombocytopenia. Immune thrombocytopenias are caused by appearance in blood of antiplatelet abtibodies and accelerated destruction of platelets sensibilized by those antibodies, and hypoproductive thrombocytopenias - by impaired platelet production in the bone marrow. Main directions of the laboratory diagnostics of thrombocytopenias - analysis of auto - and alloautoantibodies and evaluation of platelet production and turnover in the blood stream. The following methods are used for the investigation of antiplatelet antibodies: 1) measurement of platelet associated immunoglobulins; 2) determination of circulating antibodies reacting with platelets; 3) determination of antibodies using antigen specific methods - by their reactivity with isolated platelet antigens (glycoproteins). Efficacy of platelet production could be assessed by measuring in blood the amount of “young” (reticulated) platelets. One more method for the evaluation of platelet production as well as the rate of platelet turnover - measurement of plasma soluble glycocalicin, glycoprotein Ib fragment shed from the surface of platelets upon their destruction in spleen and liver. In patients with immune thrombocytopenia autoantibodies are evaluated in all cases, the percentage of reticulated platelets is significantly increased and the amount of plasma glycocalicin is within the normal range or increased. In patients with hypoproductive thrombocytopenia autoantibodies are not detected or detected at low level, the percentage of reticulated platelets is within the normal range or slightly increased and the amount of plasma glycocalicin is lowered. Diagnostics of hapten forms of immune thromocytopenias (heparin-induced thrombocytopenia and others) and of alloimmune thrombocytopenias (neonatal alloimmune thrombocytopenia in particular) are considered in the separate sections of this review

Maternal incompatibilities with fetal human platelet alloantigens -1a, -1b and -15 are the main causes of neonatal alloimmune thrombocytopenia in Russia

The aim. Mechanisms underlying the development of neonatal alloimmune thrombocytopenia (NAIT) in in Russia have been studied. Materials and methods. Genetic polymorphisms of human platelet alloantigens (HPA) -1, -2, -3, -4, -5, and -15 were evaluated in 27 families having the newborns with NAIT. NAIT was diagnosed according to the following criteria: (1) newborn with thrombocytopenia; (2) mother with no thrombocytopenia and no increase of platelet associated IgG, (3) presence of antibodies reacting with paternal platelets in maternal plasma / serum. HPA genotyping revealed incompatibilities in 23 out of 27 tested families. In these 23 families HPA-1 conflicts were detected in 16 ones (70%). In 8 cases mothers were homozygous carriers of rare HPA-1b allele and in another 8 cases - of HPA-1a allele which cased incompatibilities with fetal HPA-1a and HPA-1b respectively. In 5 out of 23 families (22%) there were incompatibilities with fetal HPA-15 (HPA-15a, n=2 and HPA-15b, n=3), in 1 family - with HPA-5b (4%), and in 1 family - with HPA-3b (4%) alloantigens. In conclusion the main causes of NAIT in Russia were HPA-1a and -1b conflicts and HPA-15 conflicts were the second frequent ones

Ретикулярные тромбоциты – новый фактор риска атеротромбоза?

In this review we described the properties of reticulated platelets (RP) and showed how variations of their content might influence platelet activity, efficacy of antiplatelet drugs and the rate of thrombotic events in patients with cardiovascular diseases. RP represent a minor platelet fraction containing residual RNA from megakaryocytes. Platelets have no nucleus and do not synthesize RNA de novo, and RNA of megakaryocytic origin is destroyed during their circulation. That is why only recently produced “young” platelets contain RNA. In healthy donors RP are identified by staining with the RNA specific fluorescent dyes by flow cytofluorimetry or using standard protocols in modern flow haematological analyzers. RP content in blood reflects the level of thrombocytopoesis in the bone marrow. RP on average amounted from 3 to 10% of all platelets in the circulation depending on the method applied for their determination. RP absolute amount and/or their percentage is changed in haematological diseases associated with the alterations of megakaryocyte productive properties. RT measurements in patients with cardiovascular diseases have shown that their content is increased in acute coronary syndrome patients. RP are larger and functionally more active in comparison with not reticulated forms. They more frequently incorporate into the platelet aggregates and contain more intracellular granules. Increase of RT content in the circulation correlates with the increase of the average size and functional activity in the whole platelet population. High RP content in patients with cardiovascular diseases reduces antiaggregative effects of aspirin and P2Y12 APD receptor antagonists and increases the risk of atherothrombotic events.В обзоре рассмотрены свойства ретикулярных тромбоцитов (РТ) и показано, как как вариации их содержания могут влиять на активность тромбоцитов, действие антитромбоцитарных препаратов и частоту тромбозов у больных сердечно-сосудистыми заболеваниями (ССЗ). РТ представляют собой минорную фракцию тромбоцитов, содержащих остаточную РНК из мегакариоцитов. Тромбоциты не имеют ядра и не синтезируют РНК de novo, а РНК мегакариоцитарного происхождения разрушается в процессе их циркуляции в кровотоке. В связи с этим РНК содержит только недавно образовавшиеся, «молодые» тромбоциты. РТ определяют по окраске РНК специфическими флуоресцентными красителями с помощью проточной цитофлуориметрии или используя стандартные протоколы в современных проточных гематологических анализаторах. Содержание в крови РТ отражает уровень тромбоцитопоэза в костном мозге. У здоровых лиц, в зависимости от способа определения, РТ составляют в среднем от 3 до 10% всех циркулирующих тромбоцитов. Абсолютное количество и/или процентное содержание РТ изменяется при гематологических патологиях, ассоциированных с изменениями продуктивных свойств мегакариоцитов. Измерения РТ у больных с ССЗ показали, что их содержание повышено у больных с острым коронарным синдромом. РТ крупнее и функционально более активны, чем неретикулярные формы. Они чаще включаются в состав агрегатов и содержат больше внутриклеточных гранул. Увеличение количества РТ в кровотоке коррелирует с увеличением среднего размера и функциональной активности в общей популяции тромбоцитов. Повышенное содержание РТ у больных с ССЗ снижает антиагрегационное действие аспирина и антагонистов P2Y12 рецепторов АДФ и увеличивает риск атеротромботических событий

The Integrin Antagonist Cilengitide Activates αVβ3, Disrupts VE-Cadherin Localization at Cell Junctions and Enhances Permeability in Endothelial Cells

Cilengitide is a high-affinity cyclic pentapeptdic αV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through αVβ3/αVβ5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the β1 family, and little is known on the effect of cilengitide on endothelial cells expressing αVβ3 but adhering through β1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on αVβ3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the β1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface αVβ3, stimulated phosphorylation of FAK (Y397 and Y576/577), Src (S418) and VE-cadherin (Y658 and Y731), redistributed αVβ3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density β1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of αVβ3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent