Biochemical and immunocytochemical characterization of coronins in platelets.

Rapid reorganization of the actin cytoskeleton in response to receptor-mediated signaling cascades allows platelets to transition from a discoid shape to a flat spread shape upon adhesion to damaged vessel walls. Coronins are conserved regulators of the actin cytoskeleton turnover but they also participate in signaling events. To gain a better picture of their functions in platelets we have undertaken a biochemical and immunocytochemical investigation with a focus on Coro1. We found that class I coronins Coro1, 2 and 3 are abundant in human and mouse platelets whereas little Coro7 can be detected. Coro1 is mainly cytosolic, but a significant amount associates with membranes in an actin-independent manner and does not translocate from or to the membrane fraction upon exposure to thrombin, collagen or prostacyclin. Coro1 rapidly translocates to the Triton insoluble cytoskeleton upon platelet stimulation with thrombin or collagen. Coro1, 2 and 3 show a diffuse cytoplasmic localization with discontinuous accumulation at the cell cortex and actin nodules of human platelets, where all three coronins colocalize. Our data are consistent with a role of coronins as integrators of extracellular signals with actin remodeling and suggests a high extent of functional overlap among class I coronins in platelets.

Coronin 1 Is Required for Integrin ß2 Translocation in Platelets.

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin ß2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin ß2 translocation are specifically or predominantly dependent on coronin 1.

Ticagrelor inverse agonist activity at the P2Y12 receptor is non-reversible versus its endogenous agonist adenosine 5´-diphosphate.

BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.

Alterations in platelet proteome signature and impaired platelet integrin αIIbß3 activation in patients with COVID-19.

BACKGROUND: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. OBJECTIVES: To characterize the mechanism of altered platelet function in COVID-19 patients. METHODS: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. RESULTS: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. CONCLUSIONS: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

Rate and risk factors for surgical site infection at a tertiary care facility in Peshawar, Pakistan.

BACKGROUND: Surgical site infections (SSI) are among the most common complications in surgical patients and have serious consequences for outcomes and costs. This study aimed to determine the rates and risk factors affecting surgical site infections and their incidence at Surgical 'C' Unit, Khyber Teaching Hospital, Peshawar, Pakistan. The study was conducted to compare with rates obtained by large international multi-centre studies. METHODS: A review of all general surgical interventions involving an incision, excluding anal procedures, performed between December 2008 and March 2009 (n=269) was undertaken. Various clinical parameters were recorded. Infection rates were calculated. Data were analysed using the Fisher's exact test. RESULTS: The overall SSI rate was 9.294%, 4.88% in clean cases (C), 8.39% in clean contaminated cases (CC), and 20.45% in contaminated or dirty cases (D). There were significantly higher surgical site infection rates among patients with combined American Society of Anaesthesiologists scores II and III than those with ASA score I in clean contaminated (p=0.0007), and dirty cases (p=0.0212). There were also significantly higher surgical site infection rates among patients with combined Co-morbidity Scale score 1-6 than those with no comorbid factors in clean contaminated (p=0.0002). Surgical site infection rate was highest in gastrointestinal system surgeries. CONCLUSION: The Surgical site infections can be minimised by adopting international protocols for surveillance.

Inhibition of Arginine Methylation Impairs Platelet Function.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to arginine residues in proteins. PRMT inhibitors are novel, promising drugs against cancer that are currently in clinical trials, which include oral administration of the drugs. However, off-target activities of systemically available PRMT inhibitors have not yet been investigated. In this work, we study the relevance of arginine methylation in platelets and investigate the effect of PRMT inhibitors on platelet function and on the expression of relevant platelet receptors. We show that (1) key platelet proteins are modified by arginine methylation; (2) incubation of human platelets with PRMT inhibitors for 4 h results in impaired capacity of platelets to aggregate in response to thrombin and collagen, with IC50 values in the µM range; and (3) treatment with PRMT inhibitors leads to decreased membrane expression and reduced activation of the critical platelet integrin αIIbß3. Our contribution opens new avenues for research on arginine methylation in platelets, including the repurposing of arginine methylation inhibitors as novel antiplatelet drugs. We also recommend that current and future clinical trials with PRMT inhibitors consider any adverse effects associated with platelet inhibition of these emerging anticancer drugs.

Tetherin/BST2, a physiologically and therapeutically relevant regulator of platelet receptor signalling.

The reactivity of platelets, which play a key role in the pathogenesis of atherothrombosis, is tightly regulated. The integral membrane protein tetherin/bone marrow stromal antigen-2 (BST-2) regulates membrane organization, altering both lipid and protein distribution within the plasma membrane. Because membrane microdomains have an established role in platelet receptor biology, we sought to characterize the physiological relevance of tetherin/BST-2 in those cells. To characterize the potential importance of tetherin/BST-2 to platelet function, we used tetherin/BST-2-/- murine platelets. In the mice, we found enhanced function and signaling downstream of a subset of membrane microdomain-expressing receptors, including the P2Y12, TP thromboxane, thrombin, and GPVI receptors. Preliminary studies in humans have revealed that treatment with interferon-α (IFN-α), which upregulates platelet tetherin/BST-2 expression, also reduces adenosine diphosphate-stimulated platelet receptor function and reactivity. A more comprehensive understanding of how tetherin/BST-2 negatively regulates receptor function was provided in cell line experiments, where we focused on the therapeutically relevant P2Y12 receptor (P2Y12R). Tetherin/BST-2 expression reduced both P2Y12R activation and trafficking, which was accompanied by reduced receptor lateral mobility specifically within membrane microdomains. In fluorescence lifetime imaging-Förster resonance energy transfer (FLIM-FRET)-based experiments, agonist stimulation reduced basal association between P2Y12R and tetherin/BST-2. Notably, the glycosylphosphatidylinositol (GPI) anchor of tetherin/BST-2 was required for both receptor interaction and observed functional effects. In summary, we established, for the first time, a fundamental role of the ubiquitously expressed protein tetherin/BST-2 in negatively regulating membrane microdomain-expressed platelet receptor function.

Influence of ASA score and Charlson Comorbidity Index on the surgical site infection rates.

OBJECTIVE: To compare the frequencies of surgical site infections (SSI) in ASA class-I (American Society of Anaesthesiologists-I) with ASA class II-III and CCI-0 (Charlson Co-morbidity Index-0) with CCI 1-6 in clean (C) and clean contaminated (CC) surgeries. STUDY DESIGN: Analytical study. PLACE AND DURATION OF STUDY: This study was conducted in a General Surgical Unit of Khyber Teaching Hospital, Peshawar, from December 2008 to April 2009. METHODOLOGY: A total of 310 clean and clean contaminated general surgical interventions with pre-operative ASA score of I-III, were included in the study, excluding anal and cystoscopic procedures. On the basis of past medical record, patients were grouped into ASA-I (patients without any co-morbidity) and ASA II-III (patients with co-morbidities) on the basis of their ASA score pre-operatively. In the same way patients were divided into CCI-0 (patients without co-morbidities) and CC 1-6 (patients with co-morbidities) according to CCI score. All the patients were operated in the same environment by the same set of surgeons. Postoperatively the surgical wounds were observed for SSI by using ASEPSIS daily scoring system for one month prospectively. SSI rates in ASA-I was compared with SSI rates in ASA II-III. Similar comparison of SSI rates was performed in CCI-0 and CCI 1-6. Data was tested by using the Fisher's exact test with confidence interval of 95%. RESULTS: The overall SSI rate was 6.1% (n=19) with 4.23% (n=5) in clean cases (C) and 7.29% (n=14) in clean contaminated cases (CC). There were significantly higher surgical site infection rates among patients in ASA II-III than those with ASA-I in clean contaminated surgeries (p=0.003). There were also significantly higher surgical site infection rates among patients with CCI score 1-6 than those with CCI-0 in clean (p=0.024) and clean contaminated (p=0.002). CONCLUSION: American Society of Anaesthesiologists (ASA) score and Charlson comorbidity index (CCI) has strong influence on SSI rates in clean and clean contaminated cases. Patients' with co-morbidities undergoing clean and clean contaminated general surgical procedures have greater SSI rates than those without any co-morbidity.

Risk Factors for the Development of Functional Tricuspid Regurgitation and Their Population-Attributable Fractions.

OBJECTIVES: The objective of this study was to determine risk factors for progression to hemodynamically significant tricuspid regurgitation (TR) and the population burden attributable to these risk factors. BACKGROUND: Few data are available with regard to risk factors associated with the development of hemodynamically significant functional TR. METHODS: A total of 1,552 subjects were studied beginning with an index echocardiogram demonstrating trivial or mild TR. Risk factors for progression to moderate or severe TR were determined by using logistic regression and classification trees. Population attributable fractions were calculated for each risk factor. RESULTS: During a median follow-up time of 38 (interquartile range [IQR]: 26 to 63) months, 292 patients (18.8%) developed moderate/severe TR. Independent predictors of TR progression were age, female sex, heart failure, pacemaker electrode, atrial fibrillation (AF), and indicators of left heart disease, including left atrial (LA) enlargement, elevated pulmonary artery pressure (PAP), and left-sided valvular disease. Classification and regression tree analysis demonstrated that the strongest predictors of TR progression were PAP of ≥36 mm Hg, LA enlargement, age ≥60 years, and AF. In the absence of these 4 risk factors, progression to moderate or severe TR occurred in ∼3% of patients. Age (28.4%) and PAP (20.5%) carried the highest population-attributable fractions for TR progression. In patients with TR progression, there was a marked concomitant increase of incident cases of elevated PAP (40%); mitral and aortic valve intervention (12%); reductions in left ventricular ejection fraction (19%), and new AF (32%) (all p < 0.01). CONCLUSIONS: TR progression is determined mainly by markers of increased left-sided filling pressures (PAP and LA enlargement), AF, and age. At the population level, age and PAP are the most important contributors to the burden of significant TR. TR progression entails a marked parallel increase in the severity of left-sided heart disease.

Alterations in Platelet Alpha-Granule Secretion and Adhesion on Collagen under Flow in Mice Lacking the Atypical Rho GTPase RhoBTB3.

Typical Rho GTPases, such as Rac1, Cdc42, and RhoA, act as molecular switches regulating various aspects of platelet cytoskeleton reorganization. The loss of these enzymes results in reduced platelet functionality. Atypical Rho GTPases of the RhoBTB subfamily are characterized by divergent domain architecture. One family member, RhoBTB3, is expressed in platelets, but its function is unclear. In the present study we examined the role of RhoBTB3 in platelet function using a knockout mouse model. We found the platelet count, size, numbers of both alpha and dense granules, and surface receptor profile in these mice were comparable to wild-type mice. Deletion of Rhobtb3 had no effect on aggregation and dense granule secretion in response to a range of agonists including thrombin, collagen, and adenosine diphosphate (ADP). By contrast, alpha-granule secretion increased in mice lacking RhoBTB3 in response to thrombin, collagen related peptide (CRP) and U46619/ADP. Integrin activation and spreading on fibrinogen and collagen under static conditions were also unimpaired; however, we observed reduced platelet accrual on collagen under flow conditions. These defects did not translate into alterations in tail bleeding time. We conclude that genetic deletion of Rhobtb3 leads to subtle alterations in alpha-granule secretion and adhesion to collagen without significant effects on hemostasis in vivo.

The membrane-associated fraction of cyclase associate protein 1 translocates to the cytosol upon platelet stimulation.

Platelets undergo profound shape changes upon adhesion to damaged blood vessel walls that are mediated by reorganisation of the actin cytoskeleton in response to receptor-mediated signalling cascades. The highly conserved 56 kDa multidomain cyclase associated protein 1 (CAP1) works in concert with cofilin and profilin to modulate actin filament turnover by facilitating cofilin-mediated actin filament severing and depolymerisation and catalysing profilin-mediated regeneration of actin monomers for reutilisation in growing filaments. CAP1 is abundant in platelets but its roles remain unexplored. We report that in suspended platelets CAP1 localises predominantly at the cell cortex whereas in spread platelets it is uniformly distributed in the cytoplasm, with enrichment at the cell cortex and the periphery of actin nodules. Upon subcellular fractionation most CAP1 was found cytosolic but part associated to the membrane fraction in an actin-independent manner. Interestingly, upon stimulation with thrombin a significant proportion of the membrane-associated CAP1 translocates to the cytosol. This relocalisation was prevented by prior treatment with PGI2 or the nitric oxide donor GSNO, or by inhibition of GSK3. Our results place CAP1 at a crossroad of signalling pathways that control platelet activation by contributing to actin remodelling at the cell cortex and actin nodules during platelet spreading.

Hypertrophic obstructive cardiomyopathy-the Leipzig experience.

BACKGROUND: Management of hypertrophic obstructive cardiomyopathy (HOCM) has evolved considerably over the last fifty years, and includes medical treatment as well as septal myectomy (SM) to relief symptoms caused by left ventricular outflow tract obstruction (LVOTO). We report the Leipzig Heart Center experience in the surgical management of patients with HOCM. METHODS: We collected data from all patient treated surgically with a myectomy for LVOTO in patients with HOCM between 1997 and 2016. Patients with absent data were excluded from our analysis. All data were obtained from our surgical database and analyzed retrospectively. RESULTS: We identified 115 patients who underwent surgical treatment for HOCM, where a male:female ratio of 1.1:1, a mean age of 60.8±14.6 years and a body mass index of 27.7±4.5 were observed. The most common symptom was dyspnea (n=102). Of all patients, 50% had New York Heart Association (NYHA) III class symptoms. Arterial hypertension was the most common comorbidity (n=80). Preoperative transthoracic echocardiography showed a mean ejection fraction (EF) of 65.9%±9.7%, median septal thickness of 21.1±5.3 mm, systolic anterior motion of the mitral valve (MV) in 61.7% of patients, and mean LVOT gradient of 70.1±45.2 mmHg. Surgery resulted in a mean decrease of the septal thickness by 15±3.7 mm and LVOT gradient by 9.7±8.5 mmHg. Isolated SM was carried out on 20 patients. Postoperatively, we were able to notice a significant improvement in NYHA classification, in which patients with NYHA III benefitted most from SM. Long-term follow-up data up to 11.3±0.7 years were available for 114 patients. During follow-up, 16 patients died, and one patient was lost to follow up. CONCLUSIONS: Surgical treatment of LVOT in patients with HOCM is an effective intervention that improves cardiac function and quality of life (QoL) and is associated with a low operative risk.

Does sex affect the outcome of laparoscopic cholecystectomy? A retrospective analysis of single center experience.

INTRODUCTION: The aim of our study was to determine the effect of sex on the outcome of laparoscopic cholecystectomy in terms of operative time, conversion to open cholecystectomy, postoperative complications and mean hospital stay. METHODS: In this retrospective observational study, we analyzed the medical records of 2061 patients who underwent laparoscopic cholecystectomy in the surgical department of Khyber Teaching Hospital (Peshawar, Pakistan) between March 2008 and January 2010. χ(2) test and t-test were respectively used to analyze categorical and numerical variables. P ≤ 0.05 was considered significant. RESULTS: The study included 1772 female and 289 male patients. The mean age for male patients was 44.07 ± 11.91 years compared to 41.29 ± 12.18 years for female patients (P = 0.706). Laparoscopic cholecystectomy was successfully completed in 1996 patients. The conversion rate was higher in men (P < 0.001), and the mean operating time was longer in men (P < 0.001). Bile duct injuries occurred more frequently in men (P < 0.001). Gallbladder perforation and gallstone spillage also occurred more commonly in men (P = 0.001); similarly severe inflammation was reported more in male patients (P = 0001). There were no statistically significant differences in mean hospital stay, wound infection and port-site herniation between men and women. Multivariate regression analysis showed that the male sex is an independent risk factor for conversion to open cholecystectomy (odds ratio = 2.65, 95% confidence interval: 1.03-6.94, P = 0.041) and biliary injuries (odds ratio = 0.95, 95% confidence interval: 0.91-0.99, P-value = 0.036). CONCLUSIONS: Laparoscopic cholecystectomy is often challenging in men on account of more adhesions and inflammation. This leads to higher conversion rates and more postoperative complications. Optimized planning and a more experienced operating surgeon may help overcome these problems.

Laparoscopic versus open appendectomy: a comparison of primary outcome measures.

BACKGROUND/AIM: The aim of the study was to compare laparoscopic and open appendectomy (OA) in terms of primary outcome measures. STUDY DESIGN: A randomized controlled trial. Place and duration of the study: Khyber Teaching Hospital, Peshawar, Pakistan, February 2008 to December 2009. PATIENTS AND METHODS: A total of 160 patients were divided into two groups, A and B. Group A patients were subjected to laparoscopic appendectomy (LA), whereas Group B patients were subjected to OA. Data regarding age, gender, and primary outcome measures, such as hospital stay, operative duration, and postoperative complication, were recorded and analyzed. Percentages were calculated for categorical data, whereas numerical data were represented as mean ± SD. Chi-square test and t test were used to compare categorical and numerical variables, respectively. Probability ≤ 0.05 (P ≤ 0.05) was considered significant. RESULTS: After randomization, 72 patients in group A and 75 patients in group B were analyzed. The mean age of patients in groups A and B was 23.09 ± 8.51 and 23.12 ± 10.42 years, respectively, (P = 0.981). The mean hospital stay was 1.52 ± 0.76 days in group A and 1.70 ± 1.06 days in group B (P = 0.294). The mean operative duration in group A and B were 47.54 ± 12.82 min and 31.36 ± 11.43 min, respectively (P < 0.001). Pain (overall level) was significantly less in group A compared with group B (P = 0.004). The two groups were comparable in terms of other postoperative complications, such as hematoma (P = 0.87), paralytic ileus (P = 0.086), urinary retention (P = 0.504), and wound infection (P = 0.134). CONCLUSION: LA is an equivalent procedure and not superior to OA in terms of primary outcome measures.

Laparoscopic treatment of hepatic hydatid cyst.

OBJECTIVE: To determine the outcome of laparoscopic management of hepatic hydatid disease in terms of complications. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Surgical C Unit, Khyber Teaching Hospital, Peshawar, from February 2007 to March 2010. METHODOLOGY: All patients with 3 or less hepatic hydatid cysts who underwent laparoscopic treatment for hepatic hydatid cyst disease were included during the study period. Laparoscopic aspiration, unroofing and evacuation of the hepatic hydatid cysts was done. Clinicopathologic features, operative time, conversion to laparotomy, morbidity, mortality and recurrence rates were analysed. RESULTS: Forty three patients had laparoscopic treatment for hepatic hydatid cysts. Females were 27 (62.79%) and males were 16 (37.20%). Mean age of patients was 38.6 ± 14.03 years (range 15-64 years). Pain was the commonest presentation occurring in 34 (79.06%) and mass in 9 (20.93%). Hepatic hydatid cysts were successfully treated laparoscopically in 40 patients. Open surgery conversion was needed in 3 (6.97%) due to inadequate access. The mean duration of surgery was 46.27 ± 13.84 minutes. Complications included port-site infection in 3 (6.97%), bile leak in 4 (9.30%) and recurrence in 2 (4.65%) cases; there was no mortality in the series. CONCLUSION: Laparoscopic hepatic hydatid cyst surgery was a safe and effective method in selected patients.