Molecular Aspects of Pathophysiology of Platelet Receptors

Receptor is a dynamic instrumental surface protein that helps to interact with specific molecules to respond accordingly. Platelet is the smallest in size among the blood components, but it plays many pivotal roles to maintain hemostasis involving its surface receptors. It (platelet) has cell adhesion receptors (e.g., integrins and glycoproteins), leucine-rich repeats receptors (e.g., TLRs, glycoprotein complex, and MMPs), selectins (e.g., CLEC, P-selectin, and CD), tetraspanins (e.g., CD and LAMP), transmembrane receptors (e.g., purinergic—P2Y and P2X1), prostaglandin receptors (e.g., TxA2, PGH2, and PGI2), immunoglobulin superfamily receptors (e.g., FcRγ and FcεR), etc. on its surface. The platelet receptors (e.g., glycoproteins, protease-activated receptors, and GPCRs) during platelet activation are over expressed and their granule contents are secreted (including neurotransmitters, cytokines, and chemokines) into circulation, which are found to be correlated with different physiological conditions. Interestingly, platelets promote metastasis through circulation protecting from cytolysis and endogenous immune surveillance involving several platelets receptors. The updated knowledge about different types of platelet receptors in all probable aspects, including their inter- and intra-signaling mechanisms, are discussed with respect to not only its (platelets) receptor type but also under different pathophysiological conditions.

Polymorphism of GPIa, GPIb and GPIIIa platelet receptors genes in women with von Willebrand disease type I

Цель исследования: изучить частоту встречаемости полиморфизмов генов тромбоцитарных рецепторов GPIa, GPIb и GPIIIa у женщин с болезнью Виллебранда I типа выполнить клиниколабораторное сопоставление для выявления наличия или отсутствия корреляционной связи между исследуемыми генетическими полиморфизмами и выраженностью дефицита фактора Виллебранда и фактора VIII. Материалы и методы. В исследование включено 136 женщин в возрасте от 18 до 45 лет (в среднем 31,7 0,5 лет) с диагностированной болезнью Виллебранда I типа. Контрольную группу составили 111 здоровых женщин в возрасте от 18 до 45 лет (в среднем 30,0 0,6 лет). Исследование антигена фактора Виллебранда (vWF:Ag), ристоцетинкофакторной активности фактора Виллебранда (vWF:RCo) и активности фактора VIII (FVIII:C) проводили на автоматическом коагулометре Elite PRO (Instrumentation Laboratory, США) с использованием реагентов HemosIL (Instrumentation Laboratory, США). Молекулярногенетическое исследование полиморфизма генов тромбоцитарных рецепторов GPIIIa (1a/1b), GPIa (C807T) и GPIb (A1/A2) выполняли методом аллельспецифической полимеразной цепной реакции с детекцией продуктов амплификации методом электрофореза в полиакриламидном геле. Результаты. Частота встречаемости полиморфизмов генов тромбоцитарных рецепторов GPIIIa (1a/1b), GPIa (C807T) и GPIb (A1/A2) у пациенток с диагностированной болезнью Виллебранда I типа не имела статистически значимых различий с таковой у здоровых женщин. Исключение составил генотип 1a1b гена тромбоцитарного рецептора GPIIIa, частота встречаемости которого у пациенток с болезнью Виллебранда I типа оказалась выше по сравнению с частотой встречаемости данного генотипа в контрольной группе (23,5 и 10,8 , соответственно p 0,05). Заключение. Полиморфизм генов тромбоцитарных рецепторов GPIIIa (1a/1b), GPIa (C807T) и GPIb (A1/A2) одинаково широко встречается как у здоровых лиц, так и у женщин с болезнью Виллебранда. Имеет ли какоелибо клиническое значение выявленная нами несколько бльшая, по сравнению со здоровыми лицами, частота встречаемости генотипа 1a1b гена тромбоцитарного рецептора GPIIIa у пациенток с болезнью Виллебранда, покажут дальнейшие исследования. Aim: to study polymorphisms frequency of GPIa, GPIb and GPIIIa platelet receptors genes in women with von Willebrand disease type I to carry out clinical and laboratory comparison to reveal presence or absence of correlation relationship between the studied genetic polymorphisms and Willebrand factor and factor VIII deficiency expression. Materials and methods. The study included 136 women aged from 18 to 45 years (mean 31.7 0.5 years) with diagnosed von Willebrand disease type I. The control group consisted of 111 healthy women aged from 18 to 45 years (mean 30.0 0.6 years). The study of von Willebrand factor antigen (vWF:Ag), ristocetincofactor activity of von Willebrand factor (vWF:RCo) and factor VIII activity (FVIII:C) was performed on Elite PRO automatic coagulometer (Instrumentation Laboratory, USA) using HemosIL reagents (Instrumentation Laboratory, USA). A moleculargenetic analysis of polymorphism of GPIIIa (1a/1b), GPIa (C807T) and GPIb (A1/A2) platelet receptor genes was performed by allelespecific polymerase chain reaction with detection of amplification products using polyacrylamide gel electrophoresis. Results. Polymorphisms frequency of GPIIIa (1a/1b), GPIa (C807T) and GPIb (A1/A2) platelet receptors genes in patients with diagnosed von Willebrand disease type I was not significantly different from that in healthy women. The exception was the genotype 1a1b of platelet receptor gene GPIIIa, the frequency of which in patients with von Willebrand disease type I was higher compared with the frequency of this genotype in the control group (23.5 and 10.8, respectively p 0.05 ). Conclusion. The polymorphism of GPIIIa (1a/1b), GPIa (C807T) and GPIb (A1/A2) platelet receptors genes is equally widespread both in healthy persons and in women with von Willebrand disease. Further studies will show if higher frequency of 1a1b genotype of GPIIIa platelet receptor gene in patients with von Willebrand disease has any clinical significance.

Effect of alteplase on platelet function and receptor expression

Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined. Results This study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

Signaling Pathways of Receptors Involved in Platelet Activation andShedding of These Receptors in Stored Platelets

All cells encounter various signals coming from the surrounding environment and they need toreceive and respond to these signals in order to perform their functions. Cell surface receptorsare responsible for signal transduction .Platelets are blood cells which perform several functionsusing diverse receptors. Platelet concentrate is one of the most consumed blood products.However, due to the short lifespan of the platelets and platelets damage during storage, we faceshortage of platelet products. One of the damages that platelets undergo during storage is theloss of surface receptors. Since cell surface receptors are responsible for all cell functions, theloss of platelet receptors reduces the quality of platelet products. In this study, we reviewed theimportant receptors involved in platelet activation and their associated signaling pathways. Wealso looked at the platelet receptors that shed during storage and the causes of this incident.We found that GPIbα, P-selectin, CD40 and GPVI are platelet receptors that fall during plateletstorage at room temperature. Considering that GPVI and GPIbα are the most important receptorswhich involved in platelet activation, their shedding can cause decrease in platelet activationafter transfusion and decrease thrombus consistence. Shear stress and platelet contact with thecontainer wall are among the mechanisms discussed in this process, but studies in this area haveto be continued.

the Importance of CD36 Expression in Evaluation of Thrombotic Risk in Patients with Myeloproliferative Neoplasms

Introduction Thrombotic complications occurring in MPNs patients have been shown to be associated with increased levels of platelet-derived microparticles (MP). The JAK2 mutational status and allele burden level increase the expression of these MP. Recently, MP have been implicated as prognostic factor and a marker for the presence of acute coronary syndromes, strokes and thromboembolism. The increase in CD36- platelet receptor expression (GPIV) is shown to be due to the platelet activation mechanism. The CD36 receptor plays a role in the fixation of the platelet on phosphatidylserine and in the recruitment of the MP into the thrombi. The CD36 signalling is important for the formation of a normal thrombus or complete platelet activation. Method This retrospective study included 157 patients with MPNs, CML, HES as well as 10 controls and 21 non haematological patients with thrombosis. The group of MPNs patients included 25 patients with chronic myeloid leukaemia (CML), 5 patients with HES and 127 patients with MPNs Ph negative (essential thrombocythemia, polycythemia vera and idiopathic myelofibrosis). For all MPNS patients it was assessed mutational status- JAK, CALR and for CML patients the disease status based by BCR/ABL expression. Blood samples were taken fasting, on citrate tube and were processed less than 4 hours in the lab. Whole blood sample was used for platelet receptors expression and platelet poor plasma (PPP) obtained by centrifuged blood at 2500G was used for MP expression The platelet receptors assessed were: CD42a,CD42b, CD41, CD61 and CD36. Sample reading was performed on BD FACS Canto II platform, FACS Diva software 7. Calibration for microparticle size was performed using Mega-mix-SSC Plus (BioCytex) for diameters of 0.16 μm, 0.20 μm, 0.24 μm and 0.5 μm for MFI setting of fluorescents SPHERO Calibration Particles 8 peaks were used, compensation was done using BD Comp-Beads and titration used poor plasma in platelets from a healthy donor. The integral microparticles were defined as positive VPD450 and the subsets were defined as CD41 + / CD61 + derived microparticles, epithelial derived microparticles such as CD31 + / CD41- / CD61- / CD45- / GlyA-, microparticles derived from erythrocytes such as GlyA + / CD31- / CD41- / CD61- / CD45- and microparticles derived from leukocytes such as CD45 + / CD31- / CD41- / CD61- / GlyA-. The platelet response was assessed by PFA200 method using ADP/Col and EPI/COL cartridge. Results The expression of CD41 is lower in MPNs patients cmpared with nonhematological(NH) patients with thrombosis, p-0.005. No differences compared with healthy volunteers. There are no differences in CD61 expression between MPNs and Nhpatients with thrombosis or healthy volunteers but we obtain a lower variation coefficient for both receptors in MPNs patients compared with controls, p=0.01 suggested a higher homogenicity The CD42a expression is higher in MPNs and CML group compared with healthy volunteers, p=0.004, the variation coefficient is higher in MPNs patients-higher heterogenicity-p=0.05. There are no differences for CD42b. Conclusions The MPNs JAK positive patients have higher number of MP compared with MPNs JAK negative patients The expression of CD36 on platelet membrane and MP platelet derived, is higher in MPNs patients, especially in JAK positive group; This expression decreases in MPNs group with thrombosis. This decrease of expression could be a marker for higher risk of thrombosis? We have to check this finding in a large group of patients The CD41/CD61 and CD 42a/CD42b receptors are lower expressed in MPNs and CML groups on the platelet membrane and PM platelet derived. The low expression does not correlate with thrombosis presence or JAK2/CALR mutation The low expression of CD41/CD61 and CD42a/CD42b is positive correlated with platelet response in PFA 200 assessing Higher risk of thrombosis in MPNs patient seems to be associated with JAK2 mutation especially those with high expression of allele burden and higher expression of CD 36. Acknowledgments This research was supported by UEFSCDI Romania center grant to PN-III-P2-2.1-PED-2016-1315 project -PARTE-MPN . Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Genetic Variability of SRC Family Kinases and Its Association with Platelet Hyperreactivity and Clinical Outcomes: A Systematic Review

Background: Platelet hyperactivity has been implicated in many cardiovascular (CV) events such as ischemic stroke, myocardial infarction and CV death. Genetic variability of platelet receptors has been shown to impact Src family kinases (SFKs) activation and in turn influence platelet activation. SFKs are important signal transmitters in platelets, interacting with several receptors as GPIIB/IIIa, GPIb, PEAR 1, GPIa, GPVI, PECAM and CD148. Methods: In this review, we focused on genetic variants of platelet receptors whose signals are transmitted mainly by SFKs and may be associated with clinical manifestations of platelet hyperactivation like MI or IS. Results: The genetic variants of platelet receptors, the signals of which are transmitted by SFKs, and the associated clinical manifestations in platelet hyperactivation, have been examined. The most extensively studied receptors were glycoprotein polymorphisms. The greatest numbers of genetic variants were analyzed in GPIb. GPIIb/IIIa receptor polymorphisms were also well analyzed and many studies highlighted their associations with ischemic stroke (IS) and myocardial infarction (MI). However, there are a number of conflicting studies finding that GPIIb/IIIa receptor polymorphisms may not influence platelet hyperactivity. Moreover, variability within some other receptors like GPVI, PECAM, PEAR1, and CD148 was analyzed only in single studies. Conclusions: Src family kinases are one of the most important signal transmitters in platelets. Some receptors have well documented interactions with SFKs, while other have not been examined in humans or data about its association originated from single studies. Further studies are necessary to confirm the findings and reduce falsepositive associations.