Platelet Aggregation Studies Research Articles

Impact of Duration of Parkinsonism and Antiparkinsonian Drugs on Coagulation Parameters in Patients with Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder involving depletion of dopaminergic neurons. Pathogenetic mechanisms leading to striatonigral degeneration are debatable. Chronic inflammation is proposed as one of the mechanisms. In any disease with underlying inflammation, immune system mediators called cytokines are released, leading to a procoagulant state. Our aim was to study the coagulation parameters in patients with PD and find their correlation with disease duration and antiparkinsonian drugs. This cross-sectional study was conducted between January 2021 and December 2021 in a tertiary care center. Sixty-eight patients with PD were subcategorized based on disease duration (<5 and ≥5 years), number of antiparkinsonian drugs (one drug and ≥2 drugs), and levodopa equivalent daily dose (LEDD; <350 and ≥350 mg). Hemoglobin (Hb), prothrombin time (PT), fibrinogen, bleeding time, and platelet aggregation study with adenosine diphosphate and collagen were assessed. In our cohort of 68 patients, 53 were men and 15 were women. Mean disease duration was 5.97 ± 4.37 years. Hb was negatively associated with LEDD (P = 0.012) and duration of the disease (P = 0.02). Similarly, PT was negatively associated with disease duration (P = 0.041) and number of antiparkinsonian drugs (P = 0.03). LEDD showed a positive association with collagen-induced platelet aggregation (P = 0.03). In our study, patients using multiple antiparkinsonian drugs and higher LEDD were observed to have a prothrombotic state. Further studies are needed to confirm these findings.

Engineering Hyperechogenic Colloids with Clot-Targeting Capabilities from Platelet-Derived Membranes.

Thrombosis-related cardiovascular diseases remain the leading global cause of mortality and morbidity. In this study, we present a pioneering approach in the field of nanobiotechnology, with a focus on clinical translation, aimed at advancing early diagnosis and enhancing treatment options for thrombotic disorders. We introduce the fabrication of Platelet Membrane-Derived Bubbles (PMBs), which exhibit distinctive characteristics compared to conventional nanoparticles. These PMBs possess an average diameter of 700 nm and a negative ζ-potential, mirroring the attributes of parent platelet membranes. Utilizing diagnostic ultrasound imaging, we demonstrated the ability to visualize PMBs as hyperechogenic entities in agarose phantoms in vitro and in live mice in vivo. Furthermore, through confocal laser microscopy, we verified the retention of crucial transmembrane proteins, such as CD41 (GPIIb) and CD42 (GPIb), pivotal in conferring platelet-specific targeting functions. Importantly, our platelet aggregation studies confirmed that PMBs do not induce platelet aggregation but instead adhere to preformed platelet-rich in vitro thrombi. Overall, our work showcases the safe and precise utilization of PMBs to directly target acute thrombosis induced by laser injury in murine mesenteric veins in vivo, as visualized through intravital microscopy. In conclusion, we have successfully developed a rapid method for generating PMBs with unique ultrasound-directed and thrombus-targeting properties. These exceptional attributes of PMBs hold significant promise for advancing the field of ultrasound diagnostic thrombus imaging and clot-targeted therapy in the clinical context.

Hydroxyurea: An Old Drug in Need of New Clinical Trials in Myeloproliferative Neoplasms?

Hydroxyurea (Hu) has been a front-line therapeutic agent for myeloproliferative neoplasms (MPN) for many years and still enjoys the endorsement of experts in the field. However, several publications have reported sub-optimal response, the need for treatment interruption because of cytopenias and lack of sustained response. In all these studies, Hu was used as continuous therapy at a daily dose ranging from 500mg to 3000mg. At our Centre we have used Hu as intermittent therapy (akin to schedules used in patients with solid tumours) at 20-30mg/Kg doses, given as a single dose, twice or thrice weekly. We have consistently observed sustained responses without troublesome cytopenias. Concurrent anti-platelet therapy was given on the basis of the results of whole blood platelet aggregation studies, achieving effective thromboprophylaxis. In this report we present our experience in 118 patients treated with intermittent Hu during the past 30 years (median follow-up 8.5 yrs): polycythemia vera – 29; essential thrombocythemia – 84; primary myelofibrosis – 5. Based on the pharmacokinetics of Hu and our experience, we speculate that the efficacy of intermittent Hu therapy without troublesome myelotoxicity over long periods of time is attributable to the following: i) higher plasma level from intake of Hu as a single dose; ii) higher uptake of Hu by cells with higher mitotic activity (i.e. the abnormal clone); and iii) unhindered, normal haemopoiesis on the drug free days each week. We hope that this article will generate interest and contemplation, leading to further publications from Centres using intermittent Hu therapy and randomized clinical studies to compare the two dosage schedules (Continuous Vs Intermittent) in MPN patients.

A rare case of glanzmann thrombasthenia with concomitant factor VII deficiency.

Glanzmann thrombasthenia and clotting factor VII deficiency are rare autosomal recessive bleeding disorders. But the occurrence of both in the same person is an extremely rare phenomenon. Here, we present the case of a young female from Sindh, Pakistan that got diagnosed with Glanzmann thrombasthenia and concomitant moderate factor VII deficiency, a combination not previously reported in the country. The patient exhibited typical clinical manifestations including menorrhagia, nasal bleeds, and prolonged bleeding after minor injuries, compounded by a positive family history and consanguinity. Laboratory investigations revealed marked anemia, prolonged bleeding time, and abnormal platelet aggregation studies consistent with Glanzmann thrombasthenia. The identification of this rare combination relied on comprehensive clinical evaluation, emphasizing the importance of family history in suspected cases. Management involved platelet transfusions, tranexamic acid, and Factor VII replacement, resulting in clinical improvement.

G protein–coupled receptor kinase 5 regulates thrombin signaling in platelets

BackgroundOur prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein–coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism. ObjectivesTo determine role of platelet specific GRK5 in platelet responses to agonists and injury. MethodsPlatelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism. ResultsWe now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo. In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism. ConclusionThese data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin.

Temporal Effect of CYP3A4/5 Induction on Ticagrelor's Pharmacodynamic Effects: A Case Series.

Ticagrelor is contraindicated in combination with cytochrome P450 3A4 and 3A5 enzyme (CYP3A4/5) inducers due to increased clearance, causing diminished antiplatelet effects. The emergent nature of acute coronary syndromes (ACS) may preclude scrutinization of home medications before P2Y12 inhibitor administration. The purpose of this case series is to establish the temporal impact of CYP3A4/5 enzyme induction on ticagrelor's pharmacodynamic effect by utilizing VerifyNow platelet aggregation studies. This was a retrospective case series of three patients who were taking a CYP3A4/5-inducing medication and loaded with ticagrelor for ACS. The duration of ticagrelor's antiplatelet effect was dramatically shortened in the presence of background CYP3A4/5 induction. The offset of antiplatelet effect, defined by platelet reactivity units (PRU), was 10-24hours in the presence of CYP3A4/5 enzyme induction compared to the anticipated 36-48 hours. This was consistent across CYP3A4/5-inducing medications including carbamazepine, phenobarbital, and phenytoin. This study demonstrates rapid return of platelet function after a ticagrelor loading dose in the presence of CYP3A4/5-inducing medications. Monitoring of PRU every 6-12hours with subsequent loading with clopidogrel or prasugrel should be considered. Larger scale studies are warranted to confirm these results.

Kenaf Seed Cysteine Protease (KSCP) Inhibits the Intrinsic Pathway of the Blood Coagulation Cascade and Platelet Aggregation.

Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects. Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken. Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP. A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation. KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications.

Emergent Coronary Thrombectomy for Acute Myocardial Infarction Immediately Following Craniotomy with Tumor Resection.

The management of perioperative acute myocardial infarction (AMI) following oncologic neurosurgery requires balancing competing risks of myocardial ischemia and postoperative bleeding. There are limited human data to establish the safest timing of antiplatelet or anticoagulation therapy following neurosurgical procedures. For patients with malignancy experiencing AMI in the acute postoperative period, staged percutaneous coronary intervention (PCI) with upfront coronary aspiration thrombectomy followed by delayed completion PCI may offer an opportunity for myocardial salvage while minimizing postoperative bleeding risks. CYP2C19 genotyping and platelet aggregation studies can help confirm adequate platelet inhibition once antiplatelet therapy is resumed.

The Utility of a Coagulation Laboratory Profile in Addition to Clinical Risk Factors for Thrombosis Risk Evaluation in Patients with Myeloproliferative Neoplasms: A Pilot Study

Background: Patients (pts) with myeloproliferative neoplasms (MPN) have a significantly increased risk of arterial and venous thrombotic events (TEs), while some pts may paradoxically suffer from bleeding manifestations. Thrombosis risk evaluation in polycythemia vera (PV) and essential thrombocythemia (ET) has important implications for patient management, however, the currently used risk stratification models are extremely limited. Methods: We designed a pilot study to assess the utility of an expanded coagulation laboratory profile in addition to other clinical variables for thrombosis and bleeding risk evaluation in a heterogenous MPN patient population. Our laboratory profile included PT, aPTT, fibrinogen, D-dimer, von-Willebrand Factor (vWF) antigen and activity testing, platelet (PLT) aggregation studies and thrombin generation assay (TGA, ST-Genesia, Stago, France), along with CBC and comprehensive biochemistry studies. Blood was drawn from pts visiting the hematology clinics or day care at Hadassah Medical Center. Clinical and demographic data were collected retrospectively from electronic medical records. The study was approved by the local IRB committee. Results:74 pts were included in the study; the mean age was 59.7 years and 51% were women. Sixty percent had a diagnosis of PV, 20% had ET, 17.6% had myelofibrosis (MF) and 2.7% had MPN unclassifiable. 78% of the pts harbored a JAK2V617F mutation, 8% and 2.7% harbored CALR and MPL mutations, respectively, and 11% were negative to all three MPN driver mutations. 63% of the pts had at least one cardiovascular risk factor, most commonly hypertension (HTN) and dyslipidemia. Most of the pts were treated with antiplatelet agents (62%), anticoagulants (16%) and/or cytoreductive agents (58%). Of 74 pts, 31% had a history of TEs (20% arterial, 11% venous), 22% reported suffering from microvascular symptoms, and 20% had experienced bleeding. The high rate of bleeding manifestations in our study population suggests an underestimation of the bleeding burden in MPN, as proposed by our group previously [Rottenstreich, Eur J Intern Med 2017]. Other patient characteristics were overall comparable to MPN patient populations in other studies (Table 1). In multivariate analyses, history of TEs was significantly associated with older age, type 2 diabetes mellitus (DM2), HTN and dyslipidemia, but not with gender, MPN subtype, driver mutation status or smoking. Pts with history of TEs had significantly lower hematocrit and prolonged PT/INR, associated with a higher rate of treatment with cytoreduction and anticoagulation, respectively, in these pts. Interestingly, pts with TEs had significantly higher vWF antigen levels (p=0.007), which were independent of age, blood counts and treatments, a finding not previously reported in MPN. No association was observed between TEs and PLT or WBC counts, D-dimer levels and vWF activity (Table 2), however, vWF activity was decreased in pts with higher PLT counts. TGA was of key interest in this study. In 62 pts not receiving anticoagulation, we observed an association between prolonged lag time (suggesting decreased coagulability) and older age, male gender and (near significant trend) JAK2V617 negative status, but did not find a correlation to TEs, blood counts and other variables. PLT aggregation indices were not associated with TEs, bleeding history or blood counts. In multivariate logistic regression analyses, history of DM2 and HTN, and vWF antigen levels, showed the strongest association to TEs, which was independent of age, blood counts and all other variables. A risk-model incorporating these variables showed a sensitivity of 77% and specificity of 92% for history of TEs in our study population. Conclusions: Despite being limited by a small and heterogenous study population and retrospective data collection; these findings support the utility of a coagulation laboratory profile in the evaluation of thrombosis and bleeding risk in MPN. The potential of TGA in risk evaluation in MPN is intriguing but merits further research. The multivariate risk-model incorporating history of DM2 and HTN along with vWF antigen levels merits validation in larger studies. Based on this pilot, a prospective study following over 250 pts with PV and ET (excluding overt MF) was initiated. This study will include NGS mutational profiling and JAK2V617F allele burden testing and omit the use of PLT aggregation studies.

Cytochrome P-450 CYP2C19 genetic polymorphism and its relation with clopidogrel resistance.

To find out the prevalence of CYP2C19*2 genetic polymorphism in ischaemic heart disease patients, and to determine its relation with clopidogrel resistance in different genotype groups. The cross-sectional study was conducted from August 2015 to December 2019 at the Army Medical College, National University of Medical Sciences, Rawalpindi, Pakistan, and comprised ischaemic heart disease patients of either gender who were on clopidogrel therapy. CYP2C19*2 genotyping of all the patients was carried out through polymerase chain reaction-restriction fragment length polymorphism. Platelet aggregation analysis was done using a light transmission aggregometer. Data was analysed using SPSS 23. Of the 390 patients, 232(59.5%) were males and 158(40.5%) were females. The overall age range was 16-82 years. Clinical indications of clopidogrel were angina 198(50.8%), myocardial infarction 146(37.4%) and acute coronary syndrome 46(11.8%). CYP2C19*2 genotyping showed that 196(50.24%) patients were homozygous wild type carriers (GG or *1/*1), 159(40.8%) were heterozygous carriers (GA or *1/*2), and 35(9%) were homozygous polymorphic allele carriers (AA or *2/*2). Platelet aggregation studies showed that there were 157(80.1%) clopidogrel responders and 39(19.9%) clopidogrel-resistant patients among GG carriers, 118(74.2%) clopidogrel responders and 41(25.8%) clopidogrel-resistant among GA carriers, and 18(51.4%) clopidogrel responders and 17(48.6%) clopidogrel-resistant among AA carriers (p=0.001). Intergroup mean platelet aggregation was significantly different (p=0.025). Allelic frequency of dominant allele *1 and polymorphic variant allele *2 was 0.706(70.6%) and 0.294(29.4), respectively. Homozygous and heterozygous carriers of CYP2C19 allele *2 was found to have higher prevalence of clopidogrel resistance in the studied population.

Platelet Electron Microscopy Does Not Correlate with Bleeding Scores in a Pediatric Population

Background:Platelet electron microscopy (PEM) is the diagnostic test for delta storage pool deficiency, a bleeding disorder due to qualitative or quantitative deficiency in dense granules leading to abnormal platelet function. Many people have non-syndromic forms which are challenging to diagnose. Aims: We aimed to describe the population for whom PEM is sent and correlate PEM results with bleeding phenotype and platelet function testing. Methods:We performed a single-center, retrospective study including patients who had PEM sent between 2/1/2018 and 8/30/2022 and had a hematology encounter. Demographics, PEM results, platelet aggregation studies, von Willebrand factor (VWF) testing, coagulation studies, genetic testing, and International Society on Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) scores were collected. ISTH-BAT was calculated based on the note closest in time to PEM testing. ISTH-BAT was either retrospectively calculated from clinical documentation or extracted as calculated by the clinician during the visit. ISTH-BAT normal range was defined as &amp;lt;4 for adult males, &amp;lt;6 for adult females, and &amp;lt;3 for children (&amp;lt;18 years) (Elbatarny et al., Hemophilia 2014). Descriptive statistics were used to summarize the results. Pearson's correlation coefficient and a Student's t-test were used for normally distributed data and Spearman rank correlation for non-parametric data. Categorical variables were compared with a Pearson chi-squared test. Statistical analysis included the first PEM result. Alpha&amp;lt;0.05 was considered significant. Results: There were 92 PEM tests sent from 77 subjects (Table 1). PEM testing was completed at either University of Toledo (N=79, normal range: 4-6 dense granules per platelet [dg/pl]) or Mayo Clinic (N=13, normal range: ≥1.2 dg/pl). Forty-six (60%) subjects had abnormal PEM results. Repeat testing was sent from 15 subjects, 13 of whom had an initial abnormal reported result with 12/13 repeat tests confirmed abnormal. Two initially indeterminant results were repeated with 1 abnormal and 1 normal on repeat. The mean dense granule per platelet for the cohort was 2.68 (standard deviation [SD] 1.4) dg/pl. The mean dense granules per platelet was 3.9 dg/pl (range 1.8 - 5.47) for subjects with normal PEM results and 1.97 dg/pl (range 0 - 3.39) for those with abnormal results, p&amp;lt;0.001. Most ISTH-BATs were retrospectively calculated from clinical documentation (71/77, 92%). The mean ISTH-BAT for the cohort was 3.6 (SD 2.2) and 50/77 (65%) had an elevated ISTH-BAT score for age and gender. There was no correlation between the ISTH-BAT score and dg/pl (Pearson's r=0.17, 95% CI: -0.60 - 0.379, p=0.15), (Figure 1). There was a weak correlation between dg/pl and ISTH-BAT for female gender (Pearson's r=0.39, 95% CI: 0.105 - 0.611, p=0.009) that is not present with male gender (Pearson's r= -0.057, 95% CI: -0.404 - 0.303, p=0.759). There was no difference between the mean dense granules per platelet for those with a normal ISTH-BAT (2.5 dg/pl, 95%CI: 1.79 - 3.22) and an abnormal ISTH-BAT (2.8 dg/pl 95% CI: 2.45 - 3.10), p=0.48. Similarly, there was no association between ISTH-BAT and PEM results as a binary variable, based on reference lab normal versus abnormal result (chi-squared p=0.22). Seventy-three platelet function tests were performed in 74% (57/77) of the cohort. The majority (43/57, 75%) had only one platelet function test. Most were whole blood impedance lumi-aggregometry (WBILA) (65/73, 89%); 8 were light transmission aggregometry. The first platelet function test was abnormal in 42% (24/57) of those tested. In the 49 subjects with WBILA as their first test, the most common abnormality was ADP secretion (N=20). ADP secretion values are not correlated with dg/pl (Spearman's r=0.28, p=0.05). At least one set of VWF tests were sent in 79% of the cohort (61/77). Two subjects were diagnosed with VWD, one of whom had a reported abnormal PEM result (2.96 dg/pl) and an ISTH-BAT of 7. The other had a normal EM result (4.38 dg/pl) and an ISTH-BAT of 4. Conclusions:There was no association between platelet EM results and bleeding phenotype by ISTH-BAT in the overall pediatric cohort, however there was a weak correlation between bleeding score and dg/pl in female gender. There was also no correlation between dg/pl and ADP secretion. Future work will focus on the utility of platelet EM testing in pediatrics and identifying subpopulations who may benefit from this test.

Platelet Aggregation Studies and Coagulation Profile in Sickle Cell Disease in Symptomatic and Steady State Patients.

To determine whether there is higher degree of platelet and/ or coagulation activation in sickle cell anaemia (SS) patients in complications and with clinical risk factors. A cross sectional study was conducted at a tertiary health care centre in central India with study groups: sickle cell disease (SCD): sickle cell anaemia (SS) and sickle cell trait (AS) consisting of 100 subjects each and controls (AA) with 40 subjects. Platelet aggregation (PA) with ADP, collagen and epinephrine, PT and aPTT were performed in all subjects and PA with ristocetin in ten candidates of each group. ANOVA and student's unpaired t test were used to compare PA and coagulation profile of the three groups with respect to age groups, gender, present diagnosis, history of complications, frequency of hospital admissions (high ≥ 3/year) and frequency of blood transfusion (high > 2/year). The max PA% with ADP was significantly less in SS patients in steady state, which was even lesser in those having symptoms, complications in past/ present, high-frequency hospital admission and > 2 blood transfusions per year subgroups, as compared to all other groups and subgroups, but not consistently with collagen and epinephrine. The max PA % with ristocetin was least in SS with complications. No statistically significant difference in PT and aPTT values among the various clinical risk subgroups and groups was found. SCD patients can be monitored by using PA with ADP for their timely and better management. PA with ADP, PT and aPTT should be added to the workup of these patients for improved prognostication.

Clinical, laboratory and ultrastructural findings in patients with storage pool disease: A case series

IntroductionPlatelet storage pool diseases (SPD) are a heterogeneous group of bleeding disorders associated with defects in the storage, secretion, or release of platelet granules. Patients with SPD present with a life-long mucocutaneous bleeding diathesis. Our goal was to study the clinical, laboratory, and ultrastructural changes in platelets of patients diagnosed with SPD using a transmission electron microscope (TEM). MethodsIn this retrospective, cross-sectional cohort study, medical records of all patients referred for evaluation of a platelet function disorder were screened for an underlying diagnosis of SPD during the period 2010–2020. ResultsSixty-eight patients were identified, among whom 62 (91.2%) had a platelet function assay (PFA) study, of whom 21 (33.9%) were abnormal. Clinical, laboratory, and light transmission aggregometry (LTA) suggested that 10 (14.7%) patients had SPD; five had gray platelet syndrome (GPS), three had Hermansky Pudlak syndrome (HPS), and two had Chedia-Higashi syndrome (CHS). Most of these cases presented with mucocutaneous bleeding diathesis, but a few had oculocutaneous albinism. They were associated with variable abnormalities in the PFA and LTA studies. However, EM studies using TEM showed a reduction/absence of alpha or delta granules in GPS and HPS patients, respectively, but no abnormality in the granules of CHS patients. ConclusionsAlthough patients with SPD presented with bleeding diathesis, PFA and platelet aggregation studies were inconclusive. Abnormalities in platelet ultrastructure on EM studies demonstrated corroborative evidence for SPD with absent/reduced alpha or delta granules.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry.

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

A correlation study between mean platelet volume and platelet aggregation study in acute coronary syndrome patients.

Platelets play an important role in cardiovascular disease mainly in the development of acute thrombotic events. Elevated platelet indices have been proposed as a risk factor for acute coronary syndrome (ACS). It remains uncertain whether increased platelet indices are the result or the cause of ACS. This study aimed to correlate mean platelet volume (MPV) and platelet aggregation response to know the functional status of platelets based on their size. A total of 50 patients with ST-segment elevation ACS (STE-ACS) or non-ST-segment elevation ACS (NSTE-ACS) were included and their MPV was measured and platelet aggregometry was performed. Patients were divided into two groups, patients with MPV ≤9.1 fl as group 1 and those with MPV >9.1 fl as group 2. The mean maximum platelet aggregation response (MMPAR) with ADP, Collagen, and Epinephrine, of both the groups, were compared. MMPAR to ADP, Collagen, and Epinephrine in group 1 was 74.47%, 66.13%, and 72.9%, respectively, and in group 2, 72.94%, 59.97%, and 72.43%, respectively. There was no statistically significant difference in the MMPAR to ADP, Collagen, and Epinephrine among the two groups. Increased MPV does not indicate the platelets are hyperreactive. An increase in MPV may be because of the increased release of immature platelets from bone marrow as there is increased consumption of platelets at the site of thrombus formation in ACS.

Aqueous Extract of Malus domestica Inhibits Human Platelet Aggregation Induced by Multiple Agonists

Cardiovascular diseases such as hypertension, myocardial infarction, and atherosclerosis are the major cause of morbidity and mortality in humankind and not only afflict industrialized nations but also affect developing countries such as Pakistan. We attempted to screen one of the most common fruits, Malus domestica (MD or apple), used in Pakistan for its potential cardio-protective effects. The crude aqueous extract (MD-Aq) was prepared and investigated for inhibitory activity against several important cardiovascular drug targets. Activities of antioxidant enzymes such as glutathione peroxidase (GPx) and superoxide dismutase (SOD) were studied, as well as total antioxidant status (TAS). Calcium channel blocking activity was measured using guinea pig ileum in an isolated tissue bath setup. Platelet aggregation studies were carried out using a dual-channel aggregometer. MD-Aq showed dose-dependent improvement in the GPX activity. The highest SOD levels were observed with 10 mg/ml and were 185 U/L compared to 174 U/L for saline, which means no significant effect by MD-Aq. TAS levels were highest at 10 mg/ml of MD-Aq, reaching 1.75 mmol/L of plasma. At 10 mg/ml, close to 90% inhibition of KCl-induced opening of calcium channels was observed, compared with the effect of 1mM Verapamil. At each dose of 1, 5, and 10 mg/ml, a significantly higher arachidonic acid-induced inhibition of platelet aggregation was observed as compared to the saline effect. MD-Aq effects on platelet-activating factor-induced platelet aggregation were less potent. We concluded that MD-Aq could elevate GPX and TAS levels, is inactive at SOD, and is excellent as a calcium channel blocker, a more potent blocker of arachidonic acid-induced aggregation than platelet-activating factor-induced aggregation. These activities can help treat various cardiovascular diseases.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom.These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

ACTION OF VENOM OF VIPERA LEBETINA ON BLOOD COAGULATION in vitro

Aim. In this study we focused on the search of fibrinogen-targeted proteases in the venom of Vipera lebetina. Methods. Fractionation of the venom was performed using FPLC chromatographic system Acta Prime on Q Sepharose. Analysis of protein mixtures was performed using SDS-PAGE. Аction on blood coagulation system was analyzed using APTT assay [2]. Proteolytic action on fibrinogen and identification of protein components with fibrinolytic activity was performed using electrophoresis of mixture of fibrinogen solution (2 mg/ml) with venom`s fractions. For a comprehensive evaluation of the effect of the obtained fractions on hemostasis, an original approach with modified aggregatometry was used [3]. This approach made it possible to take into account the ability of fractions to activate platelets, initiate blood coagulation, or inhibit platelet aggregation. Hemolytic action of fractions was estimated using fresh human red cells. Amount of released hemoglobin was estimated by spectrophotometry on Optizen POP. Results. Crude venom of V. lebetina was fractionated using ion-exchange chromatography on Q Sepharose. Elution was performed using a stepwise gradient of NaCl (0.1, 0.2, 0.3, 0.5, and 0.7 M NaCl) in 0.05 M Tris-HCl buffer at pH 8.3. Fractions eluted at 0.1 and 0.2 M of NaCl contained several proteins with different molecular weights ranging from 75 kDa to low molecular weight fractions according to the SDS-PAGE. Proteins that cleave α- and β-chains of fibrinogen were found in fractions 0.1 and 0.2, indicating the presence of an enzyme with fibrinogenolytic activity in the venom of V. lebetina. The fractions 0.3, 0.5, and 0.7 did not show any significant fibrinogenolytic activity. After platelet aggregation study we concluded that fraction 0.1 contained a protein with fibrinogenolytic activity. An increase in platelet aggregation was observed for the fraction 0.2 after the addition of ADP. This may indicate the presence of an active compound that promotes platelet aggregation. Further research is necessary to determine its nature. Fractions 0.3, 0.5, and 0.7 had no effect on platelet aggregation. A decrease in blood plasma clotting time in APTT to 5 s and 7 s, compared to a control value of 70 s, was shown for fractions eluted at NaCl concentrations of 0.1 M and 0.2 M, respectively. The fractions 0.3, 0.5 had only a slight effect on reducing blood plasma clotting. A slightly increased level of hemolysis was shown in the presence of the unbound fraction and the whole venom. It can be suggested that proteins with phospholipase activity are present in the non-binded fraction. Conclusions. Thus, fibrinogen-specific proteases, hemolytic agents, activators of blood clotting were found in the venom of Vipera lebetina. Most of these compounds must to be purified and can be used for basic biochemical research.

Ex vivo STUDY OF THE ACTION OF INTEGRIN RECEPTORS ANTAGONIST FROM ECHIS MULTISQUAMATIS SNAKE VENOM ON PLATELETS OF PREGNANT WOMEN WITH COMPICATIONS DURING GESTATION

Aim. In our work, we studied platelet aggregation in blood plasma of pregnant women and estimated the possibility of ex vivo normalization of aggregation rate using a polypeptide from the Echis multisquamatus snake venom. Previous reports demonstrated that it directly interacts with glycoprotein IIb/IIIa receptors on the surface of platelets, preventing their adhesion, thereby affecting the degree of aggregation. Methods. chromatography followed by size-exclusion chromatography on Superdex 75 using the FPLC system (ӒKTA, GE Healthcare, USA). Analysis of molecular weight of protein components was performed using SDS-PAGE. The concentration of protein was measured using spectrophotometer Optizen POP (Korea) at 280 nm. The ability of obtained protein to inhibit platelet aggregation was measured directly by aggregometry. Blood samples of women with placental disfunction during pregnancy (n = 28) were kindly provided by “Perinatal Center of Kyiv”. This study was approved by the Ethics Commission of the Shupyk National Medical Academy of Postgraduate Education and the Ethics Commission of the Kyiv Perinatal Center (# 3 from 05/05/2020). Aggregation of platelet-rich plasma (PRP) induced by ADP was investigated using aggregometry on the AP 2110 (Solar, Belarus). We compared the rate of platelet aggregation in the presence vs absence of platelet aggregation inhibitor. Results. Two-step chromatography protocol allowed us to obtain the polypeptide from the venom of Echis multisquamatus that possessed the anti-aggregatory action. SDS-PAGE analysis confirmed the homogeneity of obtained polypeptide with apparent molecular weight 14 kDa that corresponds to the platelet aggregation inhibitor reported earlier. Initial studies of ADPinduced platelet aggregation allowed selecting active concentration for the effective inhibitory action as 0.02 mg/ml. Conclusions. Platelet aggregation inhibitor from Echis multisquamatis snake venom of can be assumed as the effective agent that reduce the rate of platelet aggregation. We demonstrated it efficacy in platelet rich plasma of pregnant women that had placenta dysfunction. The use of direct antagonist of platelet integrin receptors was assumed as the prospective approach for suppressing of platelet reactivity in particular during complicated pregnancy.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.