Platelet-poor Plasma Research Articles

Overall haemostatic potential (OHP) assay can risk stratify for venous thromboembolism recurrence in anticoagulated patients

Assessing the risk of recurrent venous thromboembolism (VTE), particularly when patients are anticoagulated, remains a major challenge largely due to the lack of biomarkers. Blood was sampled from adult VTE patients recruited between January 2018 and September 2020, while receiving therapeutic anticoagulation. Results were compared to 144 healthy subjects (34.7% male, median age 42 years). Overall haemostatic potential (OHP) assay, a spectrophotometric assay, was performed on platelet-poor plasma, in which fibrin formation (triggered by small amounts of thrombin (overall coagulation potential, OCP)) and fibrinolysis (by the addition of thrombin and tissue plasminogen activator (OHP)) are simultaneously measured. Results were obtained from 196 patients (52.6% male, mean age 57.1 years). Compared to healthy subjects, VTE patients displayed significantly higher OCP (39.6 vs 34.5 units, p < 0.001) and OHP (9.3 vs 6.4 units, p < 0.001) as well as lower overall fibrinolytic potential (75.6 v s81.1%, p < 0.001). All 16 VTE recurrences, including 11 unprovoked, occurred above an OCP cut-off of 40th percentile (recurrence rate 4.32/100 patient-years (100PY), 95% confidence interval (CI) 2.39–7.80, p = 0.002). Of 97 patients who subsequently discontinued anticoagulation, all unprovoked VTE recurrences (n = 9) occurred above the 40th OCP percentile (recurrence rate 9.10/100PY, 95% CI 4.74–17.49, p = 0.005) and the 40th OHP percentile (recurrence rate 8.46/100PY, 95% CI 4.40–16.25, p = 0.009). Our pilot study demonstrates that the OHP assay can detect a hypercoagulable and hypofibrinolytic state in anticoagulated VTE patients and may be able to risk stratify VTE recurrence, allowing for more individualised decision on long-term anticoagulation. Further larger prospective studies are required.

The Occurrence of Hyperactivated Platelets and Fibrinaloid Microclots in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

We have previously demonstrated that platelet-poor plasma (PPP) obtained from patients with Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is characterized by a hypercoagulable state and contains hyperactivated platelets and considerable numbers of already-formed amyloid fibrin(ogen) or fibrinaloid microclots. Due to the substantial overlap in symptoms and etiology between Long COVID/PASC and ME/CFS, we investigated whether coagulopathies reflected in Long COVID/PASC—hypercoagulability, platelet hyperactivation, and fibrinaloid microclot formation—were present in individuals with ME/CFS and gender- and age-matched healthy controls. ME/CFS samples showed significant hypercoagulability as judged by thromboelastography of both whole blood and platelet-poor plasma. The area of plasma images containing fibrinaloid microclots was commonly more than 10-fold greater in untreated PPP from individuals with ME/CFS than in that of healthy controls. A similar difference was found when the plasma samples were treated with thrombin. Using fluorescently labelled PAC-1, which recognizes glycoprotein IIb/IIIa, and CD62P, which binds P-selectin, we observed hyperactivation of platelets in ME/CFS hematocrit samples. Using a quantitative scoring system, the ME/CFS platelets were found to have a mean spreading score of 2.72 ± 1.24 vs. 1.00 (activation with pseudopodia formation) for healthy controls. We conclude that ME/CFS is accompanied by substantial and measurable changes in coagulability, platelet hyperactivation, and fibrinaloid microclot formation. However, the fibrinaloid microclot load was not as great as was previously noted in Long COVID/PASC. Fibrinaloid microclots, in particular, may contribute to many ME/CFS symptoms, such as fatigue, seen in patients with ME/CFS, via the (temporary) blockage of microcapillaries and hence ischemia. Furthermore, fibrinaloid microclots might damage the endothelium. The discovery of these biomarkers represents an important development in ME/CFS research. It also points to possible uses for treatment strategies using known drugs and/or nutraceuticals that target systemic vascular pathology and endothelial inflammation.

The expression level of mir-21-3p in platelet-rich plasma: A potential effective factor and predictive biomarker in recurrent implantation failure.

Recurrent implantation failure (RIF) is the most important complication associated with in vitro fertilization (IVF). Despite the good quality of the transferred embryo, the success rate is rather disappointing. Therefore, predictive biomarkers for implantation are critical to making decisions about transferring high-quality embryos or cryopreserving them for cycles with a higher chance of implantation. Recently, intrauterine infusion of autologous platelet-rich plasma (PRP) has been proposed to increase the endometrial receptivity in RIF patients. PRP is rich in both growth factors and microRNAs (miRNAs). We investigated the possible association of mir-21-3p, mir-21-5p, mir-494-3p, mir-145-5p, and insulin-like growth factor-I (IGF-I) levels in PRP and platelet-poor plasma (PPP) samples with the pregnancy outcomes in RIF patients. The miRNA expression level and IGF-I concentration were assessed using real-time PCR and chemiluminescence methods respectively. Mir-21-3p was upregulated in PRP samples of the pregnant group in comparison to the nonpregnant group. There was no difference in the expression of mir-21-3p in PPP samples of these groups. The concentration of IGF-I was higher in PRP and PPP samples of the nonpregnant in comparison to the pregnant group. Receiver-operating characteristic curve analysis showed that mir-21-3p can be a valuable biomarker for the prediction of pregnancy chance in RIF patients treated with PRP.

A method based on plateletpheresis to obtain functional platelet, CD3+ and CD14+ matched populations for research immunological studies.

In previous studies with peripheral blood cells, platelet factors were found to be associated with severe allergic phenotypes. A reliable method yielding highly concentrated and pure platelet samples is usually not available for immunological studies. Plateletpheresis is widely used in the clinics for donation purposes. In this study, we designed a protocol based on plateletpheresis to obtain Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP) as well as CD3+ and CD14+ cells matched samples from a waste plateletpheresis product for immunological studies. Twenty-seven subjects were voluntarily subjected to plateletpheresis. PRP, PPP and blood cell concentrate contained in a leukocyte reduction system chamber (LRSC) were obtained in this process. CD3+ and CD14+ cells were isolated from the LRSC by density-gradient centrifugation and positive magnetic bead isolation. RNA was isolated from PRP, CD3+ and CD14+ cell samples and used for transcriptomic studies by Affymetrix. PRP and PPP samples were used for platelet protein quantification by multiplex assays. A reliable high yield method to obtain matched samples of PRP, PPP, CD3+ and CD14+ from a single donor for RNA and protein analyses has been designed. The RNA quality indicators (RQI) routinely used for other cell types were not suitable for platelet RNA characterization. Despite this, the platelet RNA was valid for transcriptomic studies by Affymetrix, as platelet transcripts obtained in our previous studies were confirmed in PRP samples. Platelet samples were enriched in platelet factors as determined in protein multiplex analysis. We have developed a method that yields not only high content and pure platelet samples from a single donor but also CD3+ and CD14+ matched samples that can be used for RNA and protein analyses in immunological studies.

Synthesis, structural characterization, and evaluation of new peptidomimetic Schiff bases as potential antithrombotic agents.

New Schiff bases functionalized with amide and phenolic groups synthesized by the condensation of 2-hydroxybenzaldehyde and 2-hydroxyacetophenone with amino acid amides which in turn were prepared in two steps from N-Boc-amino acids and homoveraltrylamine through intermediate compounds N-Boc-amino acids amides. The compounds were characterized by elemental analysis, FT-IR, UV–Vis, and NMR spectroscopy. The crystal structures of three Schiff bases were determined by single crystal X-ray diffraction. There exists O–Hcdots N, N–Hcdots O, and C–Hcdots O types of hydrogen bonds and C–Hcdotsuppi secondary bonding interactions in these crystalline solids. The Schiff bases have been screened for anticoagulant and antiplatelet aggregation activities. All the compounds showed procoagulant activity which shortens the clotting time of citrated human plasma in both platelet-rich plasma and platelet-poor plasma except the derivatives of L-methionine which showed anticoagulant activity by prolonging the clotting time. In addition, the compounds derived from benzyl cysteine and phenylalanine showed adenosine diphosphate induced antiplatelet aggregation activity, whereas others did not show any role. Moreover, all these compounds revealed non-hemolytic activity with red blood cells.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00706-022-02936-6.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness – in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Patients With Multiple Myeloma Have a Disbalanced Whole Blood Thrombin Generation Profile.

BackgroundMultiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis.ObjectivesTo get better insight in the disbalanced hemostasis of MM patients.MethodsWe conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls.ResultsIn WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls.ConclusionPatients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.

In Vitro Activity of Some Medicinal Plants on Blood Coagulation.

The concern for finding natural and curative agents without adverse side effects has prompted the interest in discovering hemostatic agents from plants. Therefore, in vitro activity of Aizoon hispanicum L. (Aizoaceae), Centaurea hyalolepis Boiss. (Asteraceae), Heliotropium maris-mortui Zohary. (Boraginaceae), Parietaria judaica L. (Urticaceae), Polygonum arenarium Waldst. & Kit. (Polygonaceae), and Verbascum sinuatum L. (Scrophulariaceae) on blood coagulation was estimated by two common tests, which are the prothrombin time test (PT) and the activated partial thromboplastin time test (aPTT). The extracted powders from the plants under this study were adjusted to be 50 mg/mL. Then, in vitro effect of these extracts on the platelet poor plasma samples was measured by an automated coagulation analyzer using PT and aPTT tests. Based on the obtained results, all plant extracts affected the coagulation cascade by rising either PT or aPTT or both, except for V. sinuatum extract, which reduced only aPTT value. Moreover, the recorded PT values showed that A. hispanicum, H. maris-mortui, and P. arenarium significantly prolonged the PT (p<0.05). Additionally, the results clearly showed that V. sinuatum acted as a coagulant agent based on aPTT values, while all other plants, in contrast, acted as strong anticoagulants. Among the plant species under study, A. hispanicum, H. maris-mortui, and P. arenarium extracts prolonged both PT and aPTT significantly (p<0.05). This could be referred to their additional effect on the common pathway. However, C. hyalolepis, P. judaica, and V. sinuatum showed no significant effect on PT values (p>0.05). The positive recorded data from this research could serve as identification of new hemostatic remedies that could be used for the commercial economic purposes and for managing several cardiovascular diseases.

P1693: THROMBOMODULIN RESISTANCE: A NEW PROTHROMBOTIC PATHWAY IN COVID-19

Background: Hypercoagulability and endothelial dysfunction have been shown to be closely related and both are considered as one of the main factors influencing the severity of COVID-19 patients. Thrombomodulin, among other factors, plays a key role as an anticoagulant factor through activation of protein C and subsequent limitation of thrombin generation. The endothelium also exerts anticoagulant activity under normal conditions. However, in COVID-19 patients, especially the most severe ones, the inflammatory process has been shown to trigger the release of the cytokine ANGPT2 through the endothelium, leading to the arrest of thrombomodulin and thus contributing to the prothrombotic state. Indeed, recent studies suggest that ANGPT2 inhibition may be explored as a therapeutic target for COVID-19 patients and other hypercoagulable diseases. However, to date there are no studies evaluating the role of thrombomodulin in COVID-19 hypercoagulability. Aims: To analyze thrombomodulin resistance in hospitalized COVID-19 patients by thrombin generation test and correlation with disease severity. Methods: Forty-two hospitalized COVID-19 patients on admission were included in the study. Blood samples were anticoagulated by trisodium citrate, and immediately centrifugated for platelet poor plasma, and frozen at -80ºC until analysis. The hemostatic profile of patients was determined by routine laboratory techniques and the Quantra® Hemostasis Analyzer. Thrombomodulin resistance was assessed by comparing Endogenous Thrombin Potential (ETP) with/without thrombomodulin using GENESIA® analyzer. Student’s t-test or Mann-Whitney U-test were used for comparison of means. Correlation was evaluated by Spearman correlation coefficient. Need for mechanical ventilation, ICU admission, encephalitis, thrombosis or death were considered as adverse events. Results: 23.8% of patients (n=10) had an adverse event during hospitalization. Patients with and without adverse events showed similar ETP (1615 vs 1486; P=0.257). However, the thrombin generation study with thrombomodulin revealed a higher ETP in patients with adverse event (1237.8 vs 786.9 mA; P=0.002). Consistent with this result, these patients showed a higher resistance to thrombomodulin with a lower % inhibition of ETP compared to patients without adverse event (24.3 vs 47.6; P=0.003). In fact, 100% of patients with adverse events showed <40% inhibition of ETP with thrombomodulin (P<0.001). Patients with a poor prognosis also showed a higher thrombin-peak (352.9 vs 310.6 nM; P=0.038), with an even more notable difference in the presence of thrombomodulin (308.6 vs 197.9 nM; P=0.006). In addition, these patients had a significantly higher thrombin generation velocity (247.8 vs. 144.3 nM/min; P=0.004) and consequently a shorter time to peak (4.4 vs 5.4 min; P=0.042). Notably, lower % inhibition by thrombomodulin and higher thrombin peak correlated significantly with higher clot stiffness measured by Quantra® (R=0.372, P=0.017 and R=0.320, P=0.042, respectively). ETP in the presence of thrombomodulin proved to be a good predictor of an adverse event during admission with an AUC of 0.791 (P<0.001). Summary/Conclusion: Measurement of thrombin generation can be a powerful tool for the analysis of patients with COVID-19 on admission and thus for risk stratification. Increased thrombomodulin resistance is associated with the presence of an adverse event in patients and may be considered as new independent prognostic marker.

Release of MMP-2 in the circulation of patients with acute coronary syndromes undergoing percutaneous coronary intervention: Role of platelets

IntroductionMatrix metalloproteinases (MMPs) of atherosclerotic tissue contribute to plaque rupture triggering acute coronary syndromes (ACS). Several MMPs, including MMP-2, are also contained in platelets and released upon activation. An increase in circulating levels of MMP-2 has been reported in patients undergoing percutaneous coronary interventions (PCI), but its time-course and origin remain unclear. Aims of our study were to assess the time-course of MMP-2 release in blood of stable and unstable coronary artery disease patients undergoing PCI and to unravel the possible contribution of platelets to its release. MethodsPeripheral blood samples were drawn immediately before, 4 and 24 h after PCI from patients with ACS (NSTEMI or STEMI, n = 21) or with stable angina (SA, n = 21). Platelet-poor plasma and washed platelet lysates were prepared and stored for subsequent assay of MMP-2 and β-thromboglobulin (β-TG), a platelet-specific protein released upon activation. ResultsPlasma MMP-2 and β-TG increased significantly 4 h after PCI and returned to baseline at 24 h in ACS patients, while they did not change in SA patients. Platelet content of MMP-2 and β-TG decreased significantly 4 h after PCI in patients with ACS, compatible with intravascular platelet activation and release, while they did not change in patients with SA. ConclusionsPCI triggers the release of MMP-2 in the circulation of ACS patients but not in that of patients with SA. Platelets activated by PCI contribute to the increase of plasma MMP-2 releasing their MMP-2 content. Given that previous mechanicistic studies have shown that MMP-2 may sustain platelet activation and unstabilize downstream-located plaques and in the long term favour restenosis and atherosclerosis progression, these data may encourage the search for therapeutic agents blocking MMP-2 release or activity in ACS.

Plasma-based assays distinguish hyperfibrinolysis and shutdown subgroups in trauma-induced coagulopathy.

Trauma patients with abnormal fibrinolysis have increased morbidity and mortality. Knowledge of mechanisms differentiating fibrinolytic phenotypes is important to optimize treatment. We hypothesized that subjects with abnormal fibrinolysis identified by whole blood viscoelastometry can also be distinguished by plasma thrombin generation, clot structure, fibrin formation, and plasmin generation measurements. Platelet-poor plasma (PPP) from an observational cross-sectional trauma cohort with fibrinolysis shutdown (% lysis at 30 minutes [LY30] < 0.9, n = 11) or hyperfibrinolysis (LY30 > 3%, n = 9) defined by whole blood thromboelastography were studied. Noninjured control subjects provided comparative samples. Thrombin generation, fibrin structure and formation, and plasmin generation were measured by fluorescence, confocal microscopy, turbidity, and a fluorescence-calibrated plasmin assay, respectively, in the absence/presence of tissue factor or tissue plasminogen activator (tPA). Whereas spontaneous thrombin generation was not detected in PPP from control subjects, PPP from hyperfibrinolysis or shutdown patients demonstrated spontaneous thrombin generation, and the lag time was shorter in hyperfibrinolysis versus shutdown. Addition of tissue factor masked this difference but revealed increased thrombin generation in hyperfibrinolysis samples. Compared with shutdown, hyperfibrinolysis PPP formed denser fibrin networks. In the absence of tPA, the fibrin formation rate was faster in shutdown than hyperfibrinolysis, but hyperfibrinolysis clots lysed spontaneously; these differences were masked by addition of tPA. Tissue plasminogen activator-stimulated plasmin generation was similar in hyperfibrinolysis and shutdown samples. Differences in LY30, fibrin structure, and lysis correlated with pH. This exploratory study using PPP-based assays identified differences in thrombin generation, fibrin formation and structure, and lysis in hyperfibrinolysis and shutdown subgroups. These groups did not differ in their ability to promote tPA-triggered plasmin generation. The ability to characterize these activities in PPP facilitates studies to identify mechanisms that promote adverse outcomes in trauma. Prognostic/Epidemiological; Level III.

The Influence of Sustained Mercury Exposure on Prothrombin Time and Partial Thromboplastin Time among Sudanese Gold Mining Workers

Background: Gold mining is the world's leading source of anthropogenic mercury pollution, negatively impacting not only miners but also the surrounding inhabitants; it has many effects on human health, especially cardiovascular problems, which lead to coagulation disorders and an increase in morbidity and mortality rate. The present study aimed to determine the prothrombin time (PT) and partial thromboplastin time (PTT) among Sudanese gold mining workers exposed to mercury. Methods and Results: This cross-sectional study was carried out among mining workers in the Red Sea state. A total of 50 mining workers were enrolled in the case group, and 50 non-mining apparently healthy subjects were the control group. About 5 ml of whole blood samples were collected in 3.2% sodium citrate blood collection tubes. Platelet poor plasma (PPP) for prothrombin time (PT) and partial thromboplastin time (PTT) measurements was obtained by using high-speed centrifugation. PT and PTT tests were performed using a standard method (Practical-Haemostasis.com.) with a Helena C2 coagulometer (Germany) and reagents manufactured by the Bio-med trademark (China). The mean age of miners was 33.5±11.5 years and occupation time - 1.94±2.1 years. The mean value of PTT was greater in the case group than in the control group (42.43±6.18 sec vs. 37.76±5.33 sec, P=0.000). In the age subgroup &lt;40 years, the PT level was longer than in the age subgroup &gt;40 years: 14.04±1.38 sec vs. 13.15±1.35 sec (P=0.045), respectively. The correlation analysis revealed a significant, direct correlation between PTT and occupation time (r=0.357, P=0.011). Conclusion: Prolonged coagulation time, notably PTT, has been revealed among mining workers, implying that these workers may have a clinically silent state of coagulation abnormalities.

Factor XIII-A Val34Leu and Tyr204Phe variants influence clot kinetics in a cohort of South African type 2 diabetes mellitus patients

Factor XIII, a transglutaminase that plays a crucial role in clot formation, consists of subunits A and B. Single nucleotide polymorphisms in Factor XIII-A have been linked to thrombotic risk. In Type 2 Diabetes mellitus (T2DM), a hypercoagulable state is thought to contribute to the high mortality rate associated with thrombotic diseases. Due to the lack of prevalence data of FXIII-A single nucleotide polymorphisms (SNPs) in T2DM in a South African cohort, this study assessed the prevalence FXIII-A Val34Leu (rs5985) and Tyr204Phe (rs3024477) SNP’s and the effect on clot kinetics in T2DM. Materials and methodsA cohort of T2DM patients (n = 100) and race, age and gender matched healthy controls (n = 101) were recruited following ethical approval. Thromboelastography® (TEG®) was used to assess the viscoelastic properties in platelet poor plasma (PPP) in controls (n = 91) and T2DM patients (n = 91) younger than 50 years old. Genomic DNA was isolated from whole blood using the Quick-DNA™ Miniprep Plus Kit and PCR-RFLP was used to genotype each sample for FXIII-A rs5985 and rs3024477 SNPs. ResultsTEG® analyses indicated a longer R-time (p < 0.0001) and higher TMRTG (p < 0.0001) in PPP of T2DM patients. Control and T2DM genotype distribution conformed to Hardy-Weinberg equilibrium (p > 0.05). There was a higher prevalence of the wildtype genotype of FXIII-A Tyr204Phe (rs3024477) SNP in T2DM (OR = 0.23, 95% CI = 0.12–0.42, p < 0.0001). The 204Phe variant was more frequent in the Caucasians (OR = 0.39, 95% CI = 0.05–0.33, p < 0.0001). The presence of the 204Phe variant in T2DM affected TMRTG (p = 0.0207). The variant affected R time (p = 0.0432) and TMRTG (p = 0.0209 and p = 0.0207) in controls and T2DM, respectively. ConclusionAn inverse association with T2DM and FXIII-A Tyr204Phe was found. A hypo coagulable PPP clot profile was observed in T2DM. A shorter reaction time was observed and but faster rate at which the clot reached maximum strength in both controls and T2DM in the presence of the 204Phe variant.

Novel Mathematical Modelling of Platelet-Poor Plasma Arising in a Blood Coagulation System with the Fractional Caputo–Fabrizio Derivative

This study develops a fractional model using the Caputo–Fabrizio derivative with order α for platelet-poor plasma arising in a blood coagulation system. The existence of solutions ensures that there are solutions to the considered system of equations. Approximate solutions to the recommended model are presented by selecting different numbers of fractional orders and initial conditions (ICs). For each case, graphs of solutions are supplied through different dimensions.

Platelet-Mediated Transfer of Cardioprotection by Remote Ischemic Conditioning and Its Abrogation by Aspirin But Not by Ticagrelor

PurposeThe role of platelets during myocardial ischemia/reperfusion (I/R) is ambivalent. They contribute to injury but also to cardioprotection. Repeated blood flow restriction and reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury and attenuate platelet activation. Whether or not platelets mediate RIC’s cardioprotective signal is currently unclear.Methods and ResultsVenous blood from healthy volunteers (without or with pretreatment of 500/1000 mg aspirin or 180 mg ticagrelor orally, 2–3 h before the study, n = 18 each) was collected before and after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/5 min deflation). Washed platelets were isolated. Platelet-poor plasma was used to prepare plasma-dialysates. Platelets (25 × 103/µL) or plasma-dialysates (1:10) prepared before and after RIC from untreated versus aspirin- or ticagrelor-pretreated volunteers, respectively, were infused into isolated buffer-perfused rat hearts. Hearts were subjected to global 30 min/120 min I/R. Infarct size was stained. Infarct size was less with infusion of platelets/plasma-dialysate after RIC (18 ± 7%/23 ± 9% of ventricular mass) than with platelets/plasma-dialysate before RIC (34 ± 7%/33 ± 8%). Aspirin pretreatment abrogated the transfer of RIC’s cardioprotection by platelets (after/before RIC, 34 ± 7%/33 ± 7%) but only attenuated that by plasma-dialysate (after/before RIC, 26 ± 8%/32 ± 5%). Ticagrelor pretreatment induced an in vivo formation of cardioprotective factor(s) per se (platelets/plasma-dialysate before RIC, 26 ± 7%/26 ± 7%) but did not impact on RIC’s cardioprotection by platelets/plasma-dialysate (20 ± 7%/21 ± 5%).ConclusionPlatelets serve as carriers for RIC’s cardioprotective signal through an aspirin-sensitive and thus cyclooxygenase-dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a humoral cardioprotective signal.Graphical abstract

Therapeutic Effect of Platelet-Rich Plasma Improves Bladder Overactivity in the Pathogenesis of Ketamine-Induced Ulcerative Cystitis in a Rat Model.

The present study attempted to elucidate whether intravesical instillation of platelet-rich plasma (PRP) could decrease bladder inflammation and ameliorate bladder hyperactivity in ketamine ulcerative cystitis (KIC) rat model. Female Sprague Dawley (S-D) rats were randomly divided into control group, ketamine-treated group, ketamine with PRP treated group, and ketamine with platelet-poor plasma (PPP) treated group. Cystometry and micturition frequency/volume studies were performed to investigate bladder function. The morphological change of bladder was investigated by Mason’s trichrome staining. Western blotting analysis were carried out to examine the protein expressions of inflammation, urothelial differentiation, proliferation, urothelial barrier function, angiogenesis and neurogenesis related proteins. The results revealed that treatment with ketamine significantly deteriorated bladder capacity, decreased voiding function and enhanced bladder overactivity. These pathological damage and interstitial fibrosis may via NF-κB/COX-2 signaling pathways and muscarinic receptor overexpression. PRP treatment decreased inflammatory fibrotic biosynthesis, attenuated oxidative stress, promoted urothelial cell regeneration, and enhanced angiogenesis and neurogenesis, thereafter recovered bladder dysfunction and ameliorate the bladder hyperactivity in KIC rat model. These findings suggested that the PRP therapy may offer new treatment options for those clinical KIC patients.

Deranged Haemostasis: Rifampin-Induced Coagulopathy

Prothrombin time (PT), first used in 1935, is the most commonly employed marker of coagulopathy to assess bleeding tendency. It is a screening test and measures the time in seconds to the formation of a clot after platelet-poor plasma has been mixed with a thromboplastin reagent consisting of tissue factor, lipids and calcium chloride ions. The International Normalized Ratio (INR) is a standardized number based on the PT of the patient. It is used by clinicians to measure the effectiveness of anticoagulants and to guide therapy. Drugs such as warfarin can affect the level of or the metabolism of vitamin K in the body, which can impact PT and INR levels. A supratherapeutic INR is rare in a patient not on therapy with vitamin K inhibitors and with preserved liver function. Here we describe an elderly man with a supratherapeutic INR, secondary to the use of rifampin. Antibiotic-induced coagulopathy is an extremely rare complication. To the authors’ knowledge, this is a unique case of rifampin-induced coagulopathy with no other associated complications of disseminated intravascular coagulation. A literature review revealed a small number of cases of rifampicin-induced coagulopathy. A high index of suspicion and knowledge is essential for the diagnosis and management of such cases.LEARNING POINTSGiven the unpredictability and rarity of rifampicin-induced coagulopathy in clinical practice, clinicians should be aware of the possibility of bleeding or drug-induced thromboembolic events with antibiotics.Early recognition and withdrawal of the offending agent are key to the successful management of this condition.Clinicians should be aware of the possible side effects of administered drugs and their possible interactions.

Human umbilical cord blood-derived platelet -rich plasma: a new window for motor function recovery and axonal regeneration after spinal cord injury

BackgroundThere are complex mechanisms for reducing intrinsic repairability and neuronal regeneration following spinal cord injury (SCI). Platelet-rich plasma (PRP) is a rich source of growth factors and has been used to motivate the regeneration of peripheral nerves in neurodegenerative disorders. However, only a few studies have shown the effects of PRP on the SCI models. MethodsWe investigated whether PRP derived from human umbilical cord blood (HUCB-PRP) could recover motor function in animals with spinal cord injury. Sixty adult male Wistar rats were randomly divided into 6 groups (n=60) as control, sham (laminectomy without induction of spinal cord injury), SCI, vehicle (SCI+ Platelet-Poor Plasma), PRP2day (SCI+PRP injection 2 days after SCI), and PRP14day (SCI+PRP injection 14 days after SCI). SCI was performed at the T12-T13 level. BBB test was carried out weekly after injury for six weeks. Caspase3 expression was determined using the Immunohistochemistry technique. The expression of GSK3β, CSF-tau, and MAG was determined using the Western blot technique. Data were analyzed by PRISM & SPSS software. ResultsHUCB-PRP treated animals showed a higher locomotor function recovery than those in the SCI group (p<0.0001). The level of caspase3, GSK3β and CSF- Tau reduced and the MAG level in the spinal cord increased by the injection of HUCB-PRP in SCI animals. ConclusionInjection of HUCB-PRP enhanced hind limb locomotor performance by modulation of caspase3, GSK3β, CSF-tau, and MAG expression. Using HUCB-PRP could be a new therapeutic option for recovering motor function and axonal regeneration after SCI.

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing.

We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.