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

Stimulation of platelet glycoprotein IIb-IIIa (αIIbβ3-integrin) functional activity by a monoclonal antibody to the N-terminal region of glycoprotein IIIa

AbstractPlatelet glycoprotein (GP) IIb-IIIa complex (αIIbβ3-integrin) changes its conformation upon platelet activation that results in binding of RGD-containing ligands and expression of ligand-induced binding site (LIBS) neoepitopes. Anti-GIIb-IIIa monoclonal antibody (monAB) CRC54 bound to ≤10% of GPIIb-IIIa on resting platelets but binding was enhanced by the occupation of GPIIb-IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N-terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb-IIIa with its ligands (fibrinogen and fibronectin) and conformation-dependent antibodies. The results indicated that changes of GPIIb-IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti-LIBS antibody with the N-terminal part of GPIIIa

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

Platelet reticulated forms, size indexes and functional activity. Interactions in healthy volunteers.

Reticulated platelets (RP) are young, functionally active platelet forms which are detected by RNA staining. Their content in the circulation reflects the intensity of bone marrow thrombocytopoesis. The aim of this study was to assess in healthy volunteers the relationship between RP percentage and platelet size and activity. RP were quantitated by thiazole orange staining using flow cytometry. Platelet size indexes included mean platelet volume (MPV), platelet large cell ratio (P-LCR) measured in a Coulter type hematological analyzer and forward scattering (FSC) measured in a flow cytometer. Platelet functional activity was evaluated by expression of activated glycoprotein (GP) IIb-IIIa (PAC-1 antibody binding) and P-selectin with the use of flow cytometry. Platelets were activated by thrombin receptor activating peptide (TRAP) (10 and 1 µM) and ADP (20 and 2.5 µM). The percentage of RP in healthy volunteers varied from 2.9% to 23.8% (mean ± SD ‒ 11.7 ± 4.7%, n = 99) and correlated with all platelet size indexes: MPV, P-LCR and FCS (r from 0.452 to 0.529, p < .001, n = 87–99). On average, RP were distributed at a ratio of 9:1 between 50% subpopulations of large and small platelets according to their FSC index. Expression of GP IIb-IIIa activated form correlated with RP percentage and platelet size indexes when platelets were activated by TRAP and ADP at both applied concentrations (r from 0.309 to 0.560, p from 0.014 to < 0.001, n = 50–62). P-selectin expression correlated with RP percentage and platelet size indexes when platelets were activated by 10 µM TRAP inducing maximum expression of this activation marker (r from 0.332 to 0.556, p from 0.008 to < 0.001, n = 65), but not by weaker agonists: 1 µM TRAP, 20 and 2.5 µM ADP (r < 0.3, n = 54–66). Thus, high RP content in healthy volunteers is associated with increased platelet size and activity in the whole platelet population

Effects of platelets activated by different agonists on fibrin formation and thrombin generation

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface

Circulating antiplatelet antibodies in pregnant women with immune thrombocytopenic purpura as predictors of thrombocytopenia in the newborns

Newborns from mothers with immune thrombocytopenic purpura (ITP) have a risk of thrombocytopenia due to passage of maternal antiplatelet antibodies into fetal/neonatal circulation. We looked for predictors of neonatal thrombocytopenia (nTP) in pregnant women with ITP. One hundred pregnant women with platelet count <100 × 109/l, no non-immune causes of thrombocytopenia and increased platelet associated IgG (PA-IgG) were included in the study. Thirty seven and 63 of them gave birth to babies with and without nTP, respectively (nTP+ and nTP− groups). Platelet count, mean platelet volume, PA-IgG, antiplatelet circulating antibodies (cAB), time of ITP onset (before or during pregnancy), and frequency of corticosteroid treatment were compared in these groups. There were no differences in all test parameters between nTP+ and nTP− groups except cAB. These antibodies were detected in 33 out of 37 in nTP+ group and in 2 out of 63 mothers in nTP− group (p < 0.001). The sensitivity of this test was 89% and its specificity was 97%. A strong reverse correlation (r = −0.749, p < 0.001) was established between maternal cAB titer and neonatal platelet count. Antibodies against glycoproteins IIb–IIIa and/or Ib were identified in antigen specific MAIPA (Monoclonal Antibody Immobilization of Platelet Antigen) assay only in 10 out of 19 (53%) test sera with cAB. Antiplatelet cAB in pregnant women with ITP could serve as reliable predictors of nTP in their babies