Altered Platelet Function Research Articles

Study of Platelet Indices in Type 2 Diabetic Patients and Its Correlation With Vascular Complications

BACKGROUND: Diabetes Mellitus is a metabolic syndrome characterized by hyperglycemia resulting in macrovascular and microvascular complications. Altered platelet morphology and functions have been linked with the pathological processes and high risk of vascular disease. Platelet indices (Mean platelet volume-MPV, Platelet distribution width-PDW and Platelet large cell ratio-PLCR) are determinants of platelet functionality. METHODS: The aim of this study was t o study the platelet indices (MPV, PDW, P-LCR) in type - 2 diabetic patients with vascular complications and compare them in diabetic patients without vascular complications. The present study was conducted on 70 diabetic patients with vascular complications and 70 diabetic patients without vascular complications for a period of one year and eight months in department of pathology, JSS hospital, Mysore. Patients were divided into cases and controls depending on the presence or absence of macrovascular complications (Myocardial infarction , stroke, peripheral arterial disease) and microvascular complications (retinopathy, nephropathy and neuropathy). Platelet indices (MPV, PDW, P-LCR) were measured using an Automated Blood Counter. FBS and HbA1C levels were collected from the clinical proforma. RESULT: Platelet indices were significantly higher in diabetic patients with vascular complications compared to those without complications [11.37 ± 1.19 fL vs 10.17 ±0 .71 fL (P=0.0001), 13.90 ± 2.99 fL vs 11.28 ± 1.55 fL (P=0.0001), 35.61 ± 9.35% vs 26.14 ± 5.79% (P=0.0001) respectively]. CONCLUSION: The present study showed a significantly higher MPV, PDW and P-LCR in diabetic patients with vascular complications compared to those without complications. This indicates that elevated platelet indices could be the cause for vascular complications. Hence MPV, PDW, P-LCR can be used as simple and cost effective predictive parameters of platelet activation to monitor and predict the risk of vascular complications. DOI: 10.21276/APALM.1297

Study of Mean platelet volume in type 2 diabetes mellitus

Background: Altered platelet morphology and function have been reported in patients with diabetes mellitus. They are likely to be associated with the pathological processes and increased risk of vascular disease seen in these patients. We aimed to determine the mean platelet volume (MPV) in diabetics compared to nonediabetics, to see if there is a difference in MPV between diabetics with and without macro and microvascular complications, and to determine the correlation between MPVand fasting blood glucose, glycosylated hemoglobin (HbA1c), patient age, and duration of diabetes, respectively. Methods: We measured MPV in 100 consecutive Type 2 diabetic patients and 50 non diabetic control subjects without known coronary artery disease who had complete blood count, FBG, HbA1c, serum creatinine and ALB/Creat ratio. Statistical evaluation was performed by SPSS for Windows statistics programme using multivariate logistical regression analysis. Results: MPV was significantly higher in diabetics compared to age- and sex-matched nonediabetic healthy controls, significantly higher with complicated DM compared to none complicated DM, and significantly higher with poor glycemic control compared to good glycemic control. Conclusions: Our results show significantly higher MPV in diabetic patients than in the nonediabetic controls. This suggests that platelets may play a role in the micro and macrovascular complications of diabetic patients.

The Potential Role of Senescence As a Modulator of Platelets and Tumorigenesis.

In addition to thrombus formation, alterations in platelet function are frequently observed in cancer patients. Importantly, both thrombus and tumor formation are influenced by age, although the mechanisms through which physiological aging modulates these processes remain poorly understood. In this context, the potential effects of senescent cells on platelet function represent pathophysiological mechanisms that deserve further exploration. Cellular senescence has traditionally been viewed as a barrier to tumorigenesis. However, far from being passive bystanders, senescent cells are metabolically active and able to secrete a variety of soluble and insoluble factors. This feature, known as the senescence-associated secretory phenotype (SASP), may provide senescent cells with the capacity to modify the tissue environment and, paradoxically, promote proliferation and neoplastic transformation of neighboring cells. In fact, the SASP-dependent ability of senescent cells to enhance tumorigenesis has been confirmed in cellular systems involving epithelial cells and fibroblasts, leaving open the question as to whether similar interactions can be extended to other cellular contexts. In this review, we discuss the diverse functions of platelets in tumorigenesis and suggest the possibility that senescent cells might also influence tumorigenesis through their ability to modulate the functional status of platelets through the SASP.

Estimation of platelet distribution width in type 2 diabetic male subjects

The patient with diabetes mellitus (DM) and its complication is increasing in our country and all over the world. Altered platelet morphology and function have been reported in patient with DM. Platelet indices act as a newly emerging and independent risk marker for atherothrombosis and cardiovascular diseases. The present study was carried out to assess Activated Partial Thromboplastin Time in subjects with type 2 diabetes mellitus. It was a cross sectional study and conducted in the Department of Physiology, Dhaka Medical College, Dhaka from July 2013 to June 2014. A total number of 200 adult male subjects were selected with age ranging from 40 to 60 years. Among them, 100 type 2 diabetic subjects were included in the study group (Group B) and 100 healthy subjects were considered as controls (Group A) for comparison. The subjects were selected from BIRDEM, Dhaka and also by personal contact from different areas of Dhaka city on the basis of exclusion and inclusion criteria. Platelet distribution width was estimated in the Department of Hematology BIRDEM, Dhaka. For statistical analysis unpaired Student’s ‘t’ test was performed. In this study, platelet distribution width was significantly (P<0.001) increased in the study group than that of control group. From this study, it may be concluded that estimation of platelet distribution width might be beneficial for prediction of future cardiovascular risk in adult diabetic male.Bangladesh Med J. 2016 Sep; 45 (3): 160-163

The Effects of 2'-O-Methoxyethyl Containing Antisense Oligonucleotides on Platelets in Human Clinical Trials.

A thorough analysis of clinical trial data in the Ionis integrated safety database (ISDB) was performed to determine if there is a class effect on platelet numbers and function in subjects treated with 2′-O-methoxyethyl (2′MOE)-modified antisense oligonucleotides (ASOs). The Ionis ISDB includes over 2,600 human subjects treated with 16 different 2′MOE ASOs in placebo-controlled and open-label clinical trials over a range of doses up to 624 mg/week and treatment durations as long as 4.6 years. This analysis showed that there is no class generic effect on platelet numbers and no incidence of confirmed platelet levels below 50 K/μL in subjects treated with 2′MOE ASOs. Only 7 of 2,638 (0.3%) subjects treated with a 2′MOE ASO experienced a confirmed postbaseline (BSLN) platelet count between 100 and 50 K/μL. Three of sixteen 2′MOE ASOs had >10% incidence of platelet decreases >30% from BSLN, suggesting that certain sequences may associate with clinically insignificant platelet declines. Further to these results, we found no evidence that 2′MOE ASOs alter platelet function, as measured by the lack of clinically relevant bleeding in the presence or absence of other drugs that alter platelet function and/or number and by the results from trials conducted with the factor XI (FXI) ASO.

189 Altered platelet function and thrombosis in fetal growth restriction

BackgroundFetal growth restriction (FGR) affects 3%–8% of pregnancies and is associated with increased risk of stillbirth and perinatal morbidity, in addition to increased risk of cardiovascular disease later in life....

Predictive role of neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio in normal-tension glaucoma

As a type of primary open-angle glaucoma, normal-tension glaucoma (NTG) is a health problem that manifests itself with low intraocular pressure (IOP) levels and preserves its importance for public health care. In population-based screenings, which take the IOP levels of 21mmHg as a cut-off for the diagnosis of glaucoma, the diagnosis is usually delayed, and the treatment is started in an advanced stage by the referred ophthalmologists. NTG is associated with some autoimmune conditions and systemic diseases, such as paraproteinemia, nocturnal hypotension, and migraine. Recently, a simple and cheap test (namely the hemogram) was defined as measure of systemic inflammation, measuring the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). A recent study displayed an altered platelet function in NTG patients. We hypothesize that an automated hemogram analysis for NLR together with IOP measurements may be used to screen NTG patients who are admitted to primary care clinics if any relationship between NTG and NLR could be demonstrated in future researches.

Polymorphisms of Platelet Glycoprotein Receptors and Cell Adhesion Molecules in Fetuses with Fetal Growth Restriction and Their Mothers As Detected with Pyrosequencing.

Vascular thrombotic tendency may lead to fetal growth restriction (FGR). Altered platelet function and genetic heterogeneity may play a role in this procedure. We investigated whether maternal or fetal genotypic frequencies of genes polymorphisms for certain platelet receptor and cell adhesion molecules are altered in FGR. We compared the maternal and fetal genotypic frequencies of single nucleotide polymorphisms (SNPs) in four genes coding for platelet receptors and cell adhesion molecules [integrin alpha subunit 2 (ITGA2)C807T, integrin subunit beta 3(ITGB3) T1565C, platelet cell adhesion protein 1 (PECAM1) CTG-GTG and selectin P(SELP)A/C]. A total of 32 fetuses with fetal growth restriction and their mothers were matched with 18 normal controls. Using maternal venous blood and umbilical cord blood samples, nucleotide sequences were determined from pyrograms. Genotypic frequencies were calculated and analyzed using appropriate tests and logistic regression. There was no statistical difference in the proportion of heterozygotes or homozygotes for any of the genotypic frequencies between FGR and control groups in mothers or fetuses. Our study demonstrated no association of maternal or fetal ITGA2 C807T SNP, ITGB3 T1565C SNP, PECAM1 CTG - GTG and SELP A/C polymorphisms with FGR.

Tamoxifen Directly Inhibits Platelet Angiogenic Potential and Platelet-Mediated Metastasis.

Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment of breast cancer. Tamoxifen and its metabolites have been shown to directly impact platelet function, suggesting that this drug has additional mechanisms of action. The purpose of this study was to determine whether tamoxifen exerts antitumor effects through direct platelet inhibition. This study found that pretreatment with tamoxifen leads to a significant inhibition of platelet activation. Platelets exposed to tamoxifen released significantly lower amounts of proangiogenic regulator vascular endothelial growth factor. In vitro angiogenesis assays confirmed that tamoxifen pretreatment led to diminished capillary tube formation and decreased endothelial migration. Tamoxifen and its metabolite, 4-hydroxytamoxifen, also significantly inhibited the ability of platelets to promote metastasis in vitro. Using a membrane-based array, we identified several proteins associated with angiogenesis metastasis that were lower in activated releasate from tamoxifen-treated platelets, including angiogenin, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 5, epidermal growth factor, chemokine (C-X-C motif) ligand 5, platelet-derived growth factor dimeric isoform BB, whereas antiangiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished vascular endothelial growth factor release, and lower angiogenic and metastatic potential. We demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen directly alter platelet function leading to decreased angiogenic and metastatic potential. Furthermore, this study supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in angiogenesis and malignancy.

Glycated albumin modifies platelet adhesion and aggregation responses

A diabetic vasculature is detrimental to cardiovascular health through the actions of advanced glycation end products (AGEs) on endothelial cells and platelets. Platelets activated by AGEs agonize endothelial responses promoting cardiovascular disease (CVD) development. While it has been established that AGEs can alter platelet functions, little is known about the specific platelet pathways that AGEs modify. Therefore, we evaluated the effects of AGEs on specific salient platelet pathways related to CVDs and whether the effects that AGEs elicit are dependent on glycation extent. To accomplish our objective, platelets were incubated with reversibly or irreversibly glycated albumin. A time course for adhesion and aggregation agonist receptor expression was assessed. Optical platelet aggregometry was used to confirm the functional activity of platelets after AGE exposure. In general, platelets subjected to glycated albumin had a significantly enhanced adhesion and aggregation potential. Furthermore, we observed an enhancement in dense body secretion and intracellular calcium concentration. This was especially prevalent for platelets exposed to irreversibly glycated albumin. Additionally, functional aggregation correlated well with receptor expression, suggesting that AGE-induced altered receptor sensitivity translated to altered platelet functions. Our findings indicate that under diabetic vascular conditions platelets become more susceptible to activation and aggregation due to an overall enhanced receptor expression, which may act to promote CVD development.

Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy.

IntroductionThe aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen.Materials and methodsVenous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed.ResultsNo statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%.ConclusionsBlood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced.

Evaluation of role of antiplatelet therapy on bleeding after dental extraction

Aims and Objectives: Antiplatelet therapy results in altered platelet function and increased bleeding time (BT). The present study was done to evaluate antiplatelet therapy on bleeding after dental extraction. Materials and Methods: In this prospective case–control study, a total of 150 patients requiring dental extraction were divided into two equal groups; Group I: with 75 patients on antiplatelet therapy and Group II: with 75 patients who have discontinued antiplatelet therapy 5 days before the dental extraction. Informed consent was obtained from the participants, and ethical approval was obtained from the Institutional Ethical Committee. Prior to extraction, BT of all the participants was recorded. Simple extraction for single-molar tooth was done in both the groups under local anesthesia. Suturing followed by pressure pack was placed after extraction. BT after extraction was statistically compared between the groups after 1 h and 24 h using Chi-square test with P < 0.05. Results: None of the patients in both the groups showed active bleeding 1 h and 24 h postoperatively. No bleeding was seen in 71 patients in Group I and 74 patients in Group II after 24 h. Conclusion: The present study showed no significant difference in BT in both the groups. Hence, single-tooth extraction can be done in patients on long-term antiplatelet therapy without discontinuation or alteration of medication.

Comparison of the effects of a balanced crystalloid-based and a saline-based tetrastarch solution on canine whole blood coagulation and platelet function.

To evaluate the effects of a 6% hydroxyethyl starch (130/0.42) solution in either a buffered, electrolyte-balanced (HES-BAL), or a saline (HES-SAL) carrier solution on canine platelet function and whole blood coagulation. Prospective, randomized study. University teaching hospital. Thirty-seven client-owned dogs undergoing general anesthesia for arthroscopy or imaging studies. Dogs received a 15 mL/kg intravenous bolus of HES-SAL (n = 13), HES-BAL (n = 14), or a modified Ringer's solution (n = 10) over 30-40 minutes. Coagulation was analyzed using a Platelet Function Analyzer-100 (closure time [CtPFA ]), and whole blood thromboelastometry (ROTEM) with extrinsically (ex-tem and fib-tem) and intrinsically (in-tem) activated assays, which assessed clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF), and lysis index (LI). Coagulation samples were assayed prior to fluid administration (T0), and 5 minutes (T1), and 3 hours (T2) following fluid bolus administration, respectively. Both HES solutions resulted in impaired platelet function as indicated by a significant prolongation of CtPFA at T1 as compared to T0, but which resolved by T2. An IV bolus of Ringer's solution did not alter platelet function. In both HES groups, whole blood coagulation was significantly impaired at T1 as indicated by a significant increase in in-tem CFT, and a significant decrease in ex-tem, in-tem, and fib-tem MCF compared to T0. Furthermore, a significant increase in ex-tem CFT at T1 compared to T0 was found in the HES-SAL group. With the exception of in-tem MCF after HES-BAL, these effects were not present at T2. No significant differences were found in CtPFA or any ROTEM variable at any time point between HES-SAL and HES-BAL. Administration of a single bolus of 15 mL/kg 6% HES 130/0.42 results in significant but short-lived impairment of canine platelet function and whole blood coagulation, regardless of carrier solution.

Analysis of Primary Hemostasis Alterations Induced By Ibrutinib Assessed By Shutter Speed and Aggregometry Impedance

Introduction:Ibrutinib is a covalent inhibitor of Bruton Tyrosine Kinase (BTK), approved for the treatment of Mantle Cell Lymphoma (MCL), Chronic Lymphocytic Leukemia (CLL) and Waldenstrom's Macroglobulinemia (WM). BTK is expressed in platelets and is believed to have a central role in platelet activation through GPIb and GPIV pathway. However, the clinical significance of inhibition of BTK is unclear in terms of bleeding and it is known that patients with congenitally deficient BTK do not exhibit increased risk of bleeding.Objectives:The main objectives were: (1) identify possible alterations in hemostasis induced by Ibrutinib in patients who start taking the drug, and (2) to monitor the development of hemorrhagic adverse effects in patients who start taking Ibrutinib.Methods:From August 31, 2015 and January 31, 2016 we conducted a prospective study including all consecutive patients with the diagnosis of MCL, CLL or WM treated in 2nd line or more, with Ibrutinib as single-drug therapy. The following parameters were analyzed on day 0 (baseline), 10 and 28 after starting treatment with Ibrutinib: APTT, PT, platelet count, PFA-100 Collagen/Epinephrine and Collagen/ADP, aggregometry impedance in whole blood by multiplate assay with AA, ADP, Ristocetin and TRAP as agonists. Von Willebrand factor antigen (vWF:Ag), Ristocetin cofactor (vWF:RCo) and coagulant factor VIII were also analyzed. We collected prospectively bleeding adverse events.Results:In the period of study, we enrolled 11 patients, 7 were males and 4 females, with a median age of 63 years old (range, 53-88). Five patients had the diagnosis of CLL, 5 patients had MCL and 1 patient had WM. After a median follow-up of 6 months, 3 out of 11 (27.3%) developed grade 1 bleeding adverse events. Of these, 1 patient was under anticoagulant treatment with intermediate dose of enoxaparin, and another patient had low levels of vWF:Ag and vWF:RCo, suggesting a possible acquired von Willebrand disease. Five out of 11 (45.5%) of patients had a prolonged shutter speed Collagen/Epinephrine at baseline, with a median of 175 sec (range, 94-270). After Ibrutinib exposure, median shutter speed Collagen/epinephrine at 10 days was 231 sec (range, 108-287), p=0.02; and at 30 days was 142 sec (range, 79-300), p=n.s. Four out of 11 patients (36.4%) had a prolonged shutter speed Collagen/ADP at base line, with a median of 105 sec (range, 63-190). After Ibrutinib exposure, median shutter speed Collagen/ADP at 10 days was 107 sec (range, 66-192), p=n.s; and at 30 days was 78 sec (range, 75-149), p=n.s. Regarding platelet aggregometry impedance, 2 patients had abnormal platelet aggregation baseline at TRAP assay, 6 at ADP assay, 3 at AA assay and 4 at Ristocetin assay. After Ibrutinib exposure, there was a worsening of platelet aggregation in ADP analysis at 10 days of treatment but became normal after 30 days of treatment. There was an improvement with TRAP at both 10 and 30 days of treatment. Of notice, all 3 patients with minor bleeding had abnormal hemostasis studies baseline before starting with Ibrutinib.Conclusions:Almost half of patients showed an altered haemostasis baseline studies before starting with Ibrutinib. The biology of the disease appears to cause in subjects primary hemostasis changes that result in a basal bleeding risk regardless of the treatment used. Only minor bleeding complications were observed under Ibrutinib treatment, all with baseline altered platelet function; 2 of the three patients who developed bleeding complications had other bleeding risk factors associated. PFA-100 Collagen/Epinephrine is prolonged in almost 50% of patients after Ibrutinib is started compared with baseline study. Aggregometry impedance only worsened with ADP at 10 days, but went into normal level at 30 days. There is an interindividual variability in alterations of haemostasis and hemorrhagic clinical signs. DisclosuresCordoba:Janssen: Research Funding, Speakers Bureau.

Pro-Inflammatory Cytokines Drive the Development of a Prothrombotic Platelet Phenotype in Inflammation and Aging

A high incidence of thrombotic events remains a potentially life-threatening consequence in a broad range of chronic inflammatory conditions including infection, obesity, cardiovascular disease (CVD), autoimmune disorders and aging. While deregulation of pro-coagulant molecules and activation of endothelial surfaces have been described as contributors to such thrombotic events, the extent to which the pro-inflammatory cytokine environment associated with inflammatory diseases may impact platelet reactivity, production and function remains to be further characterized. Hence, to directly interrogate the impact of inflammation on platelet function, we analyzed the platelet activation profile of washed platelets by flow cytometry using murine models of rheumatoid arthritis, collagen-induced arthritis (CIA) and of aging (24 month-old mice) to identify inflammation-induced changes in platelet function and production. Notably, both models revealed significant alterations to platelet function, characterized by increased exposure of P-selectin and the active form of the integrin aIIbb3 (JON/A) following platelet activation with thrombin. In addition, the exposure of phosphatidylserine (PS), measured with Lactadherin binding, was elevated in platelets from chronically inflamed CIA and old mice.As the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor α (TNF) are key mediators of chronic inflammatory disease, we assessed using Tnf ΔARE transgenic mice, which overexpress TNF-α as well as young mice treated with daily injections of rIL1- for 20 days, whether chronic exposure these cytokines is sufficient to alter platelet function. Strikingly, despite increased peripheral blood platelet numbers, IL-1β treated mice did not exhibit a hyper-reactive platelet phenotype when compared to controls. On the other hand, platelets from Tnf ΔARE mice showed significant hyperactivity, and a single dose (24 hours) of murine rTNF or in vitro culture with rTNF also induced a robust platelet activation profile, suggesting that TNF acts directly on platelets and may play a more significant role than IL-1 in the development of an inflammation-driven prothrombotic platelet phenotype.Moreover, analysis of the hematopoietic stem cell (HSC) compartment in CIA and old mice revealed an increased frequency of previously-described CD41+ megakaryocyte-biased HSCs, which is recapitulated in mice chronically exposed to IL-1, suggesting that reprogramming of HSCs by pro-inflammatory cytokines likely fuels the overproduction of hyperactive platelets that contribute to thrombosis in chronic inflammatory diseases. Collectively, our findings suggest that IL-1 and TNFα contribute to inflammation-associated thrombosis via distinct mechanisms, with TNFα playing a particularly important role in platelet hyperactivation, and IL-1 impacting platelet production, likely in part via modulation of HSC fate. Hence, our findings identify a key role for distinct cytokine circuits in regulating platelet function, and implicate the pro-inflammatory cytokine environment as a key therapeutic target for modulation in patients at risk for inflammation-associated thrombotic events. DisclosuresDi Paola:CSL BEhring: Consultancy; Biogen: Consultancy.

A study of the possible association of plasminogen activator inhibitor type 1 4G/5G insertion/deletion polymorphism with susceptibility to schizophrenia and in its subtypes.

Inhibition of the fibrinolytic system may occur at the level of plasminogen activation, mainly by PAI-1. Mental and physical stress caused to alterations of platelet function, and also decreased to fibrinolytic activity. Furthermore, stress-induced thrombosis regulation was proposed to be by PAI-1 in schizophrenia patients. In this study, the distribution of genotypes and frequency of alleles of the plasminogen activator inhibitor type 1 (PAI-1) gene 4G/5G polymorphism in different Turkish clinical schizophrenia subtypes was investigated for its role in schizophrenia development. The clinical schizophrenia subtypes include paranoid, catatonic, disorganized, undifferentiated and residual, as diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition IV (DSM-IV). Samples of genomic DNA (250 total, including 150 schizophrenia patients and 100 healthy subjects) were analysed. PAI-1 4G/5G genotyping was performed by polymerase chain reaction-allele-specific amplification. PCR products were separated by 2% agarose gel electrophoresis and then visualized. The genotype distributions (P = 0·136) and allele frequencies (P = 0·721 for 4G, P = 0. 097 for 5G) were not significantly different between patients with schizophrenia and control subjects for the 4G/5G polymorphism. Similar results were also found for the genotype distributions (P = 0·640) and allele frequencies (P = 0·763 for 4G, P = 0·448 for 5G) in the clinical schizophrenia subtypes compared to the each other. We conclude that PAI-1 4G/5G polymorphism was not significantly associated with schizophrenia or its subtypes in the Turkish population. However, we recognize that with our sample sizes, we cannot exclude weak associations.

RGDfK-functionalized gold nanorods bind only to activated platelets.

Integrin-targeting peptide RGDfK-labeled gold nanorods (GNR) seek to improve hyperthermia targeted to solid tumors by exploiting the known up-regulation of integrin αvβ3 cell membrane proteins on solid tumor vasculature surfaces. Tumor binding specificity might be expected since surrounding tissues and endothelial cells have limited numbers of these receptors. However, RGD peptide binding to many proteins is promiscuous, with known affinity to several families of cell integrin receptors, and also possible binding to platelets after intravenous infusion via a different integrin receptor, αIIbβ3, on platelets. Binding of RGDfK-targeted GNR could considerably impact platelet function, ultimately leading to increased risk of bleeding or thrombosis depending on the degree of interaction. We sought to determine if RGDfK-labeled GNR could interact with platelets and alter platelet function. Targeted and untargeted nanorods exhibited little interaction with resting platelets in platelet rich plasma (PRP) preparations. However, upon platelet activation, peptide-targeted nanorods bound actively to platelets. Addition of RGDfK-GNR to unactivated platelets had little effect on markers of platelet activation, indicating that RGDfK-nanorods were incapable of inducing platelet activation. We next tested whether activated platelet function was altered in the presence of peptide-targeted nanorods. Platelet aggregation in whole blood and PRP in the presence of targeted nanorods had no significant effect on platelet aggregation. These data suggest that RGDfK-GNR alone have little impact on platelet function in plasma. However, nonspecific nanorod binding may occur in vascular beds where activated platelets are normally cleared, such as the spleen and liver, producing a possible toxicity risk for these nanomaterials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 209-217, 2017.

Platelet Hyperactivity in TNFSF14/LIGHT Knockout Mouse Model of Impaired Healing.

Objective: Impaired and chronic wounds occur due to defects in one or more of the overlapping stages of healing. However, problems related to the vascular system are critical for nonhealing, and chronic wounds in humans often show the presence of fibrin cuffs/clots. We hypothesized that these clots are due to alterations in platelet function; hence, we have investigated whether alterations in platelet function are present during impaired healing. Approach: Platelets were subjected to different agonists to determine the rate of aggregation and evaluate the molecules involved in adhesion and aggregation that could lead to faster thrombosis and potentially contribute to impaired wound healing. Results: We show that wounding of TNFSF14/LIGHT-/- mice, which have impaired healing, leads to an enhanced response in platelet aggregation and a faster time to blood vessel occlusion (thrombosis). In addition, after wounding, platelets from these mice have increased levels of P-selectin, integrin αIIbβ3, and phosphatidylserine, molecules that contribute to platelet adhesion. They also have more extensive open canalicular system than platelets of control mice, suggesting increased surface area for interactions upon activation. Innovation: These results show a novel function for TNFSF14/LIGHT during wound healing. Conclusion: The absence of TNFSF14/LIGHT from the cell surface of platelets causes rapid platelet aggregation and thrombus formation that may contribute to impaired healing by reducing the ability of the blood vessels to transport nutrients and oxygen and other molecules needed for proper healing.

Activation of the protein C pathway and endothelial glycocalyx shedding is associated with coagulopathy in an ovine model of trauma and hemorrhage.

Acute traumatic coagulopathy (ATC) is an endogenous coagulopathy that develops following tissue injury and shock. The pathogenesis of ATC remains poorly understood, with platelet dysfunction, activation of the protein C pathway, and endothelial glycocalyx shedding all hypothesized to contribute to onset. The primary aim of this study was to develop an ovine model of traumatic coagulopathy, with a secondary aim of assessing proposed pathophysiological mechanisms within this model. Twelve adult Samm-Border Leicester cross ewes were anesthetized, instrumented, and divided into three groups. The moderate trauma group (n = 4) underwent 20% blood volume hemorrhage, bilateral tibial fractures, and pulmonary contusions. The severe trauma group (n = 4) underwent the same injuries, an additional hamstring crush injury, and 30% blood volume hemorrhage. The remaining animals (n = 4) were uninjured controls. Blood samples were collected at baseline and regularly after injury for evaluation of routine hematology, arterial blood gases, coagulation and platelet function, and factor V, factor VIII, plasminogen activator inhibitor 1, syndecan 1, and hyaluranon levels. At 4 hours after injury, a mean increase in international normalized ratio of 20.50% ± 12.16% was evident in the severe trauma group and 22.50% ± 1.00% in the moderate trauma group. An increase in activated partial thromboplastin time was evident in both groups, with a mean of 34.25 ± 1.71 seconds evident at 2 hours in the severe trauma animals and 34.75 ± 2.50 seconds evident at 4 hours in the moderate trauma animals. This was accompanied by a reduction in ROTEM EXTEM A10 in the severe trauma group to 40.75 ± 8.42 mm at 3 hours after injury. Arterial lactate and indices of coagulation function were significantly correlated (R = -0.86, p < 0.0001). Coagulopathy was also correlated with activation of the protein C pathway and endothelial glycocalyx shedding. While a significant reduction in platelet count was evident in the severe trauma group at 30 minutes after injury (p = 0.018), there was no evidence of altered platelet function on induced aggregation testing. Significant fibrinolysis was not evident. Animals in the severe trauma group developed coagulation changes consistent with current definitions of ATC. The degree of coagulopathy was correlated with the degree of shock, quantified by arterial lactate. Activation of the protein C pathway and endothelial glycocalyx shedding were correlated with the development of coagulopathy; however, altered platelet function was not evident in this model.

Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome

The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.