Publications by authors named "Emanuela Falcinelli"

51 Publications

The ISTH bleeding assessment tool as predictor of bleeding events in inherited platelet disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

J Thromb Haemost 2021 May;19(5):1364-1371

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study).

Objectives: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients.

Methods: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded.

Results: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population.

Conclusions: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.
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http://dx.doi.org/10.1111/jth.15263DOI Listing
May 2021

Walking-induced endothelial dysfunction predicts ischemic cardiovascular events in patients with intermittent claudication.

Vasc Med 2021 Apr 12:1358863X211001927. Epub 2021 Apr 12.

Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

Endothelial dysfunction, evaluated by flow-mediated dilatation (FMD), predicts adverse cardiovascular events in patients with intermittent claudication (IC). IC is an example of repeated ischemia/reperfusion injury that may contribute to the progression of vascular disease by worsening endothelial function, a trigger for acute cardiovascular events. The predictive value of effort-induced endothelial dysfunction for cardiovascular events in patients with IC has not been studied previously. The objective of this study was to assess whether exercise-induced endothelial dysfunction is predictive of adverse cardiovascular outcome in IC. In 44 patients with IC, we measured brachial artery FMD by B-mode ultrasonography at rest and 10 minutes after a maximal treadmill exercise. Treadmill exercise halved the FMD (from 3.5 ± 0.6% to 1.45 ± 0.46%, < 0.05). After a follow-up period of 85 (72-98) months, a total of 20 major cardiovascular events occurred. In a multivariate analysis, a post-exercise reduction of brachial FMD > 1.3% was predictive for cardiovascular events. Maximal exercise-induced endothelial dysfunction is predictive of cardiovascular events in patients with IC.
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http://dx.doi.org/10.1177/1358863X211001927DOI Listing
April 2021

Effect of First Long-Term Training on Whole Blood Count and Blood Clotting Parameters in Thoroughbreds.

Animals (Basel) 2021 Feb 9;11(2). Epub 2021 Feb 9.

Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy.

Training has a strong effect on the physiology of hematological parameters and blood coagulation, both in humans and in horses. Several blood changes have been reported after exercise in horses but available data differ. We aimed to investigate modifications in complete blood count and some hemostatic parameters induced by the first training period in young untrained Thoroughbred racehorses to detect a possible labile blood coagulability in racehorses. Twenty-nine untrained 2-year-old Thoroughbreds were followed during their incremental 4-month sprint exercise schedule. Blood collection was performed once a month, five times (T-30, T0, T30, T60 and T90), before and during the training period for measurement of complete blood count (CBC) and blood clotting parameters (prothrombin time-PT, activated partial prothrombin time-APTT, thrombin clotting time-TCT, fibrinogen-Fb, thrombin-antithrombin complex-TAT). Differences among the time points for each parameter were analyzed (ANOVA, Kruskal-Wallis one-way analysis of variance, < 0.05). In Thoroughbreds, the first long-term exercise workout period was found to induce a statistical increase in red blood cell indexes and lymphocytes, eosinophils and platelet counts, as well as a hypercoagulability state evident at 30 days of training, which returned to basal levels after 90 days. Regular physical exercise seems to blunt the negative effects of acute efforts on hematological and clotting parameters, an effect that may be attributed to the training condition.
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http://dx.doi.org/10.3390/ani11020447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915801PMC
February 2021

Learning the Ropes of Platelet Count Regulation: Inherited Thrombocytopenias.

J Clin Med 2021 Feb 2;10(3). Epub 2021 Feb 2.

Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Centro Didattico, Edificio B Piano 1, 06132 Perugia, Italy.

Inherited thrombocytopenias (IT) are a group of hereditary disorders characterized by a reduced platelet count sometimes associated with abnormal platelet function, which can lead to bleeding but also to syndromic manifestations and predispositions to other disorders. Currently at least 41 disorders caused by mutations in 42 different genes have been described. The pathogenic mechanisms of many forms of IT have been identified as well as the gene variants implicated in megakaryocyte maturation or platelet formation and clearance, while for several of them the pathogenic mechanism is still unknown. A range of therapeutic approaches are now available to improve survival and quality of life of patients with IT; it is thus important to recognize an IT and establish a precise diagnosis. ITs may be difficult to diagnose and an initial accurate clinical evaluation is mandatory. A combination of clinical and traditional laboratory approaches together with advanced sequencing techniques provide the highest rate of diagnostic success. Despite advancement in the diagnosis of IT, around 50% of patients still do not receive a diagnosis, therefore further research in the field of ITs is warranted to further improve patient care.
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http://dx.doi.org/10.3390/jcm10030533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867147PMC
February 2021

Search for SARS-CoV-2 RNA in platelets from COVID-19 patients.

Platelets 2021 Feb 21;32(2):284-287. Epub 2020 Dec 21.

Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

The frequent finding of thrombocytopenia in patients with severe SARS-CoV-2 infection (COVID-19) and previous evidence that several viruses enter platelets suggest that SARS-CoV-2 might be internalized by platelets of COVID-19. Aim of our study was to assess the presence of SARS-CoV-2 RNA in platelets from hospitalized patients with aconfirmed diagnosis of COVID-19. RNA was extracted from platelets, leukocytes and serum from 24 COVID-19 patients and 3 healthy controls, real-time PCR and ddPCR for viral genes were carried out. SARS-CoV-2 RNA was not detected in any of the samples analyzed nor in healthy controls, by either RT-PCR or ddPCR, while RNA samples from nasopharyngeal swabs of COVID-19 patients were correctly identified. Viral RNA was not detected independently of viral load, of positive nasopharyngeal swabs, or viremia, the last detected in only one patient (4.1%). SARS-CoV-2 entry in platelets is not acommon phenomenon in COVID-19 patients, differently from other viral infections.
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http://dx.doi.org/10.1080/09537104.2020.1859104DOI Listing
February 2021

Comparative evaluation of the fully automated HemosIL AcuStar ADAMTS13 activity assay vs. ELISA: possible interference by autoantibodies different from anti ADAMTS-13.

Clin Chem Lab Med 2021 Apr 19;59(5):e193-e196. Epub 2020 Nov 19.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

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http://dx.doi.org/10.1515/cclm-2020-1020DOI Listing
April 2021

Association of Neutrophil Activation, More Than Platelet Activation, With Thrombotic Complications in Coronavirus Disease 2019.

J Infect Dis 2021 03;223(6):933-944

Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

Background: Severe acute respiratory syndrome coronavirus 2 infection is associated with hypercoagulability, which predisposes to venous thromboembolism (VTE). We analyzed platelet and neutrophil activation in patients with coronavirus disease 2019 (COVID-19) and their association with VTE.

Methods: Hospitalized patients with COVID-19 and age- and sex-matched healthy controls were studied. Platelet and leukocyte activation, neutrophil extracellular traps (NETs), and matrix metalloproteinase 9, a neutrophil-released enzyme, were measured. Four patients were restudied after recovery. The activating effect of plasma from patients with COVID-19 on control platelets and leukocytes and the inhibiting activity of common antithrombotic agents on it were studied.

Results: A total of 36 patients with COVID-19 and 31 healthy controls were studied; VTE developed in 8 of 36 patients with COVID-19 (22.2%). Platelets and neutrophils were activated in patients with COVID-19. NET, but not platelet activation, biomarkers correlated with disease severity and were associated with thrombosis. Plasmatic matrix metalloproteinase 9 was significantly increased in patients with COVID-19. Platelet and neutrophil activation markers, but less so NETs, normalized after recovery. In vitro, plasma from patients with COVID-19 triggered platelet and neutrophil activation and NET formation, the latter blocked by therapeutic-dose low-molecular-weight heparin, but not by aspirin or dypiridamole.

Conclusions: Platelet and neutrophil activation are key features of patients with COVID-19. NET biomarkers may help to predict clinical worsening and VTE and may guide low-molecular-weight heparin treatment.
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http://dx.doi.org/10.1093/infdis/jiaa756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798977PMC
March 2021

Validation of the ISTH/SSC bleeding assessment tool for inherited platelet disorders: A communication from the Platelet Physiology SSC.

J Thromb Haemost 2020 03 16;18(3):732-739. Epub 2019 Dec 16.

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: Careful assessment of bleeding history is the first step in the evaluation of patients with mild/moderate bleeding disorders, and the use of a bleeding assessment tool (BAT) is strongly encouraged. Although a few studies have assessed the utility of the ISTH-BAT in patients with inherited platelet function disorders (IPFD) none of them was sufficiently large to draw conclusions and/or included appropriate control groups.

Objectives: The aim of the present study was to test the utility of the ISTH-BAT in a large cohort of patients with a well-defined diagnosis of inherited platelets disorder in comparison with two parallel cohorts, one of patients with type-1 von Willebrand disease (VWD-1) and one of healthy controls (HC).

Patients/methods: We enrolled 1098 subjects, 482 of whom had inherited platelet disorders (196 IPFD and 286 inherited platelet number disorders [IT]) from 17 countries.

Results: IPFD patients had significantly higher bleeding score (BS; median 9) than VWD-1 patients (median 5), a higher number of hemorrhagic symptoms (4 versus 3), and higher percentage of patients with clinically relevant symptoms (score > 2). The ISTH-BAT showed excellent discrimination power between IPFD and HC (0.9 < area under the curve [AUC] < 1), moderate (0.7 < AUC < 0.9) between IPFD and VWD-1 and between IPFD and inherited thrombocytopenia (IT), while it was inaccurate (AUC ≤ 0.7) in discriminating IT from HC.

Conclusions: The ISTH-BAT allows to efficiently discriminate IPFD from HC, while it has lower accuracy in distinguishing IPFD from VWD-1. Therefore, the ISTH-BAT appears useful for identifying subjects requiring laboratory evaluation for a suspected IPFD once VWD is preliminarily excluded.
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http://dx.doi.org/10.1111/jth.14683DOI Listing
March 2020

Development of anti-matrix metalloproteinase-2 (MMP-2) nanobodies as potential therapeutic and diagnostic tools.

Nanomedicine 2020 02 24;24:102103. Epub 2019 Oct 24.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy. Electronic address:

Matrix metalloproteinase-2 (MMP-2) is an endopeptidase involved in cardiovascular disease and cancer. To date, no highly selective MMP-2 inhibitors have been identified for potential use in humans. Aim of our work was to apply the nanobody technology to the generation of highly selective inhibitors of human MMP-2 and to assess their effects on platelet function and their applicability as conjugated nanobodies. We constructed a nanobody library after immunising an alpaca with human active MMP-2 and identified, after phage display and screening, one MMP-2 inhibitory nanobody (VHH-29), able to hinder the effects of MMP-2 on platelet activation, and one nanobody not inhibiting MMP-2 activity (VHH-136) which, chemically conjugated to a fluorescent probe, allowed the detection of human MMP-2 by flow-cytometry and immune-cytochemistry. In conclusion, we have generated and characterized two new nanotechnological molecular tools for human MMP-2 which represent promising agents for the study of MMP-2 in cardiovascular pathophysiology.
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http://dx.doi.org/10.1016/j.nano.2019.102103DOI Listing
February 2020

Next-generation sequencing for the diagnosis of MYH9-RD: Predicting pathogenic variants.

Hum Mutat 2020 01 15;41(1):277-290. Epub 2019 Oct 15.

School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK.

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice.
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http://dx.doi.org/10.1002/humu.23927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972977PMC
January 2020

Antithrombotic prophylaxis for surgery-associated venous thromboembolism risk in patients with inherited platelet disorders. The SPATA-DVT Study.

Haematologica 2020 07 26;105(7):1948-1956. Epub 2019 Sep 26.

Department of Transfusion and Cell Transplantation Kitasato University School of Medicine, Sagamihara, Japan.

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery.
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http://dx.doi.org/10.3324/haematol.2019.227876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327644PMC
July 2020

Inherited platelet disorders in women.

Thromb Res 2019 Sep;181 Suppl 1:S54-S59

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

Inherited platelet disorders (IPD) are a heterogeneous group of hemorrhagic diseases affecting both men and women, but usually associated with more evident bleeding symptoms in women due to the exposure to sexspecific hemostatic challenges, like menstruation and delivery. Indeed, up to 50% of women presenting with menorrhagia are diagnosed an IPD, moreover women with IPD can have ovulation-associated bleeding events and are at higher risk of endometriosis. Large retrospective studies have shown that women with IPD have a significantly increased risk of post-partum hemorrhage, predicted by a high bleeding score at previous history and by a platelet count below 50X10/L. In addition, in patients with IPD, female sex was associated with a higher frequency of excessive bleeding after surgery, even when excluding gynecological procedures. In conclusion, IPD may represent a serious problem for women's health, and their diagnosis and appropriate management is crucial to ensure female patients a good quality of life.
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http://dx.doi.org/10.1016/S0049-3848(19)30368-8DOI Listing
September 2019

Maraviroc Intensification Modulates Atherosclerotic Progression in HIV-Suppressed Patients at High Cardiovascular Risk. A Randomized, Crossover Pilot Study.

Open Forum Infect Dis 2019 Apr 7;6(4):ofz112. Epub 2019 Mar 7.

Unit of Infectious Diseases, University of Perugia, Perugia, Italy.

Background: Experimental CCR5 antagonism with maraviroc in atherosclerosis-prone mice and preliminary data in humans suggest an anti-atherosclerotic effect of the drug. We assessed the impact of maraviroc treatment in persons living with HIV on subclinical indicators of atherosclerosis.

Methods: Persons living with HIV on effective antiretroviral therapy (ART) including only protease inhibitors were recruited if they had a Framingham risk score >20% and brachial flow-mediated dilation (bFMD) <4%, as indices of high cardiovascular risk. Maraviroc (300 mg per os for 24 weeks) was administered, in addition to ongoing ART, to all patients using a crossover design. Brachial FMD, carotid-femoral pulse wave velocity (cfPWV), and carotid intima-media thickness (cIMT) were measured as markers of atherosclerosis. Vascular competence-as expressed by the ratio of circulating endothelial microparticles (EMPs) to endothelial progenitor cells (EPCs)-and markers of systemic inflammation and monocyte and platelet activation were assessed.

Results: Maraviroc treatment significantly improved bFMD, cfPWV, and cIMT by 66%, 11%, and 13%, respectively ( = .002, = .022, = .038, respectively). We also found a beneficial effect of maraviroc on the EMP/EPC ratio ( < .001) and platelet/leucocyte aggregates ( = .013). No significant changes in markers of systemic inflammation, monocyte activation, and microbial translocation were observed.

Conclusions: Maraviroc led to significant improvements in several markers for cardiovascular risk, endothelial dysfunction, arterial stiffness, and early carotid atherosclerosis, which was accompanied by an increase of vascular competence, without seeming to affect systemic inflammation. Our data support the need for larger studies to test for any effects of maraviroc on preventing atherosclerosis-driven pathologies.
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http://dx.doi.org/10.1093/ofid/ofz112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446135PMC
April 2019

Inhibition of platelet function after ocular administration of non-steroidal anti-inflammatory drugs.

Thromb Res 2019 Mar 11;175:1-5. Epub 2019 Jan 11.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Italy. Electronic address:

Introduction: The use of topical NSAIDs is frequent in ophthalmology to reduce the local inflammatory reaction resulting from surgical procedures. Ocular use of some drugs was previously found to lead to significant systemic absorption with possible systemic effects. NSAIDs may enhance the hemorrhagic risk of anticoagulant and antiplatelet drugs. Aim of our study was to evaluate the systemic effects of two NSAIDs given by eyedrops on platelet COX-1 and on ex vivo and in vivo platelet activation.

Materials And Methods: 20 patients planned to undergo cataract surgery were randomized to the use of an ophthalmic solution containing Diclofenac or Indomethacin. Blood was taken at enrollment (baseline) and after 3 days of therapy (1 drop, 4 times a day). Arachidonic Acid (AA)-induced light transmission aggregometry (LTA), PFA-100® C-EPI, circulating platelet P-Selectin expression by flow cytometry and serum and AA-induced TxB production were evaluated before and after eyedrop therapy.

Results: AA (0.1-0.2 mM)-induced LTA was significantly reduced after ocular indomethacin but not after diclofenac. PFA-100® C-EPI closure time was also significantly prolonged in the indomethacin group but not in the diclofenac group. Circulating platelet P-selectin expression was significantly reduced after treatment with indomethacin compared with diclofenac. Finally, treatment with eyedrop indomethacin, but not with diclofenac, strikingly suppressed AA-induced TxB generation, while treatment with diclofenac did not modify it.

Conclusions: Our data show that indomethacin administered by ophthalmic eye drops has a relevant systemic antiplatelet effect. This should be taken into account in patients under concurrent therapy with antiplatelet or anticoagulant agents.
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http://dx.doi.org/10.1016/j.thromres.2019.01.005DOI Listing
March 2019

Platelet function assays in diagnosis: an update.

Expert Rev Hematol 2019 01 16;12(1):29-46. Epub 2019 Jan 16.

a Department of Medicine, Section of Internal and Cardiovascular Medicine , University of Perugia , Perugia , Italy.

Introduction: Hemorrhagic diseases associated with platelet dysfunction include inherited platelet function disorders (IPFD) and a large number of non-hereditary conditions, defined as acquired platelet function disorders (APFD). Their identification requires a careful clinical evaluation and a rational use of diagnostic laboratory assays. Areas covered: Here we describe the laboratory techniques currently available for the assessment of platelet function, including new and experimental laboratory assays, and their alterations in platelet function disorders. Although useful and very widely used in diagnostics, none of them replicates thoroughly the in vivo setting. Expert commentary: The goals of platelet function testing are to provide a rapid and precise diagnosis to every patient bleeding due to a platelet disorder, to assess the individual bleeding risk and potentially to monitor the efficacy of prohemostatic interventions. Most of the tests currently available do not fulfill these needs due to lack of standardization, complexity, and inability to reproduce the multiple reactions involved in the role of platelets in primary hemostasis. These goals can in perspective be achieved by a continuous effort to standardize, improve and expand platelet function assays, by the generation of standardized diagnostic algorithms and, for IPFD, by the implementation of next-generation sequencing-based methods in the diagnostic practice.
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http://dx.doi.org/10.1080/17474086.2019.1562333DOI Listing
January 2019

Effect of aspirin treatment on abacavir-associated platelet hyperreactivity in HIV-infected patients.

Int J Cardiol 2018 07 12;263:118-124. Epub 2018 Apr 12.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University Perugia, Italy. Electronic address:

Background: Ischemic cardiovascular events are a relevant cause of morbidity and mortality in HIV-infected patients. Use of abacavir (ABC), a nucleoside analog reverse transcriptase inhibitor, has been associated with increased risk of myocardial infarction (MI) and with platelet hyperreactivity. We explored whether low-dose aspirin reduces in vivo platelet activation and platelet hyperreactivity induced by ABC in HIV-infected subjects.

Methods And Results: In a randomized, placebo-controlled, cross-over study forty HIV-infected patients with ABC-associated platelet hyperreactivity, defined by a score based on laboratory variables reflecting in vivo platelet activation and ex vivo platelet hyperresponsiveness, were randomized to aspirin 100 mg daily for 15 days with subsequent cross-over to placebo for additional 15 days or placebo for 15 days with subsequent cross-over to aspirin for further 15 days. In vivo and ex vivo platelet activation markers were measured at day 15 and 30. One group of healthy subjects, one of untreated HIV infected-patients and one treated without ABC, were studied concomitantly. Serum TxB and urinary 11-dehydro-TxB were decreased by aspirin in ABC-treated patients, but not as much as in healthy controls. Aspirin therapy reduced significantly platelet hyperreactivity (score: from 9.3, 95% CIs 8.7 to 10.0, to 7.5, 6.9 to 8.0), however without bringing it back to the levels of healthy controls (score: 4.6, 95% CIs 3.6 to 5.6).

Conclusion: Aspirin reduces ABC-induced in vivo platelet activation and platelet hyperreactivity in HIV-infected patients, however without normalizing them. Whether the observed reduction of platelet activation is sufficient to prevent cardiovascular events requires a prospective trial.
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http://dx.doi.org/10.1016/j.ijcard.2018.04.052DOI Listing
July 2018

Platelets Contribute to the Accumulation of Matrix Metalloproteinase Type 2 in Synovial Fluid in Osteoarthritis.

Thromb Haemost 2017 11 30;117(11):2116-2124. Epub 2017 Nov 30.

Rheumatology Unit, Department of Medicine, University of Perugia, Perugia, Italy.

Inflammation plays a role in the initiation and progression of osteoarthritis (OA), a chronic degenerative joint disorder. Platelets are inflammatory cells, contain and release matrix metalloproteinases (MMPs) and favour the release of these enzymes, key effectors of cartilage and subchondral bone degradation, by other cells; however, their role in OA has not been investigated yet. Our aims were (1) to assess the presence of platelets and of MMP-2 in synovial fluid (SF) of OA patients; (2) to evaluate the contribution of platelets to MMP-2 release by fibroblast-like synoviocytes (FLS); and (3) to investigate if hyaluronic acid (HA) interferes with these processes. SF was collected from 27 OA patients before and after treatment with intra-articular HA (20 mg/2 mL). Moreover, FLS were co-cultured with platelets, and the release of MMP-2 in supernatants was measured. Our results show that platelets are present in OA SF and show markers of activation. OA SF also contains relevant amounts of MMP-2. Co-incubation of platelets with FLS favours the release of MMP-2 by the interaction of platelet surface P-selectin with FLS CD44 by a mechanism involving the activation of pAkt and pSrc in FLS. Administration of HA to OA patients decreased the infiltration of platelets in SF and reduced the levels of MMP-2. The addition of HA in vitro inhibited the release of MMP-2 by FLS triggered by the interaction with platelets. In conclusion, our data show that platelets may contribute to joint degeneration in OA by favouring the accumulation of MMP-2 in SF.
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http://dx.doi.org/10.1160/TH17-06-0379DOI Listing
November 2017

Increase of von Willebrand factor with aging in type 1 von Willebrand disease: fact or fiction?

Haematologica 2017 11 27;102(11):e431-e433. Epub 2017 Jul 27.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Italy

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http://dx.doi.org/10.3324/haematol.2017.168013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664402PMC
November 2017

Endothelial activation in patients with superficial vein thrombosis (SVT) of the lower limbs.

Thromb Res 2017 09 27;157:20-22. Epub 2017 Jun 27.

Section of Internal and Cardiovascular Medicine, Dept. of Internal Medicine, University of Perugia, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.thromres.2017.06.033DOI Listing
September 2017

Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice.

Blood 2017 07 13;130(4):527-536. Epub 2017 Jun 13.

Department of Biology and Biotechnology, University of Pavia, Pavia, Italy; and.

The amyloid precursor protein (APP), primarily known as the precursor of amyloid peptides that accumulate in the brain of patients with Alzheimer disease, is abundant in platelets, but its physiological function remains unknown. In this study, we investigated the role of APP in hemostasis and thrombosis, using APP knockout (KO) mice. Ex vivo aggregation, secretion, and integrin αIIbβ3 inside-out activation induced by several agonists were normal in APP-deficient platelets, but the number of circulating platelets was reduced by about 20%, and their size was slightly increased. Tail bleeding time was normal, and in vivo, the absence of APP did not alter thrombus formation in the femoral artery. In contrast, in a model of vein thrombosis induced by flow restriction in the inferior vena cava, APP-KO mice, as well as chimeric mice with selective deficiency of APP in blood cells, developed much larger thrombi than control animals, and were more sensitive to embolization. Consistent with this, in a pulmonary thromboembolism model, larger vessels were occluded. APP-KO mice displayed a shorter APTT, but not PT, when measured in the presence of platelets. Moreover, the activity of factor XIa (FXIa), but not FXIIa, was higher in APP-KO mice compared with controls. APP-KO mice presented a higher number of circulating platelet-leukocyte aggregates, and neutrophils displayed a greater tendency to protrude extracellular traps, which were more strongly incorporated into venous thrombi. These results indicate that platelet APP limits venous thromboembolism through a negative regulation of both fibrin formation and neutrophil function.
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http://dx.doi.org/10.1182/blood-2017-01-764910DOI Listing
July 2017

Matrix Metalloproteinases and Platelet Function.

Prog Mol Biol Transl Sci 2017 21;147:133-165. Epub 2017 Mar 21.

Section of Internal and Cardiovascular Medicine, University of Perugia, Perugia, Italy.

Platelets contain and release several matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, -2, -3, -9, and -14 and TIMP-1, -2, and -4. Although devoid of a nucleus, platelets also synthesize TIMP-2 upon activation. Platelet-released MMPs/TIMPs, as well as MMPs generated by other cells within the cardiovascular system, modulate platelet function in health and disease. In particular, a normal hemostatic platelet response to vessel wall injury may be transformed into pathologic thrombus formation by the release from platelets and/or by the local generation of some MMPs. Moreover, platelets may localize the production of leukocyte-derived MMPs to sites of vascular damage, contributing to atherosclerosis development and complications and to arterial aneurysm formation. Finally, the interaction between platelets and tumor cells is strongly influenced by MMPs/TIMPs. All these mechanisms are emerging as important in atherothrombosis, inflammatory disease, and cancer growth and dissemination. Increasing knowledge of these mechanisms may open the way to novel therapeutic approaches.
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http://dx.doi.org/10.1016/bs.pmbts.2017.01.002DOI Listing
July 2017

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders.

Haematologica 2017 07 6;102(7):1192-1203. Epub 2017 Apr 6.

Hematology Department, S. Bortolo Hospital, Vicenza, Italy.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.
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http://dx.doi.org/10.3324/haematol.2016.160754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566025PMC
July 2017

A novel mechanism regulating human platelet activation by MMP-2-mediated PAR1 biased signaling.

Blood 2017 02 29;129(7):883-895. Epub 2016 Dec 29.

Division of Internal and Cardiovascular Medicine, Department of Medicine, University of Perugia, Perugia, Italy; and.

Platelets contain and release several matrix metalloproteinases (MMPs). Among these, active MMP-2 enhances platelet aggregation by favoring the activation of phosphatidylinositol 3- kinase (PI3K) and contributes to arterial thrombosis. The platelet surface target of MMP-2 and the mechanism through which it primes platelets to respond to subsequent stimuli are still unknown. We show that active MMP-2 enhances platelet activation induced by weak stimuli by cleaving PAR1 at a noncanonical extracellular site different from the thrombin-cleavage site and thus initiates biased receptor signaling, triggering only some of the signaling pathways normally activated by full PAR1 agonism. The novel PAR1-tethered ligand exposed by MMP-2 stimulates PAR1-dependent G and G pathway activation, triggering p38-MAPK phosphorylation, Ca fluxes, and PI3K activation, but not G signaling; this is insufficient to cause platelet aggregation, but it is enough to predispose platelets to fully respond to G-activating stimuli. Integrin αβ is a necessary cofactor for PAR1 cleavage by MMP-2 by binding the MMP-2 hemopexin domain, thus favoring the interaction of the enzyme with PAR1. Our studies unravel a novel mechanism regulating platelet activation that involves the binding of MMP-2 to integrin αβ and the subsequent cleavage of PAR1 by active MMP-2 at a noncanonical site, exposing a previously undescribed tethered ligand that triggers biased G-protein agonism and thus predisposes platelets to full activation by other stimuli. These results identify the MMP-2-αβ-PAR1 interaction as a potential target for the prevention of arterial thrombosis.
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http://dx.doi.org/10.1182/blood-2016-06-724245DOI Listing
February 2017

Inherited platelet function disorders. Diagnostic approach and management.

Hamostaseologie 2016 Nov 3;36(4):265-278. Epub 2016 Aug 3.

Paolo Gresele, MD, PhD, Division of Internal and Cardiovascular Medicine Department of Medicine, University of Perugia, Via E. dal Pozzo, 06126 Perugia, Italy, Tel. +39/07 55 78 39 89, Fax +39/07 55 71 60 83, E-Mail:

Inherited platelet function disorders (IPFDs) make up a significant proportion of congenital bleeding diatheses, but they remain poorly understood and often difficult to diagnose. Therefore, a rational diagnostic approach, based on a standardized sequence of laboratory tests, with consecutive steps of increasing level of complexity, plays a crucial role in the diagnosis of most IPFDs. In this review we discuss a diagnostic approach through platelet phenotyping and genotyping and we give an overview of the options for the management of bleeding in these disorders and an account of the few systematic studies on the bleeding risk associated with invasive procedures and its treatment.
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http://dx.doi.org/10.5482/HAMO-16-02-0002DOI Listing
November 2016

Inherited Platelet Function Disorders: Algorithms for Phenotypic and Genetic Investigation.

Semin Thromb Hemost 2016 Apr 10;42(3):292-305. Epub 2016 Mar 10.

Division of Internal and Cardiovascular Medicine, Department of Medicine, University of Perugia, Perugia, Italy.

Inherited platelet function disorders (IPFDs) manifest with mucocutaneous bleeding and are frequently difficult to diagnose due to their heterogeneity, the complexity of the platelet activation pathways and a lack of standardization of the platelet function laboratory assays and of their use for this purpose. A rational diagnostic approach to IPFDs should follow an algorithm where clinical examination and a stepwise laboratory evaluation play a crucial role. A streamlined panel of laboratory tests, with consecutive steps of increasing level of complexity, allows the phenotypic characterization of most IPFDs. A first-line diagnosis of a significant fraction of the IPFD may be made also at nonspecialized centers by using relatively simple tests, including platelet count, peripheral blood smear, light transmission aggregometry, measurement of platelet granule content and release, and the expression of glycoproteins by flow cytometry. Some of the most complex, second- and third-step tests may be performed only in highly specialized laboratories. Genotyping, including the widespread application of next-generation sequencing, has enabled discovery in the last few years of several novel genes associated with platelet disorders and this method may eventually become a first-line diagnostic approach; however, a preliminary clinical and laboratory phenotypic characterization nowadays still remains crucial for diagnosis of IPFDs.
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http://dx.doi.org/10.1055/s-0035-1570078DOI Listing
April 2016

Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

Thromb Haemost 2016 Jan 29;115(2):333-43. Epub 2015 Oct 29.

Paolo Gresele, MD, PhD, Section of Internal and Cardiovascular Medicine, Department of Medicine, University of Perugia, Via E. Dal Pozzo, 06126 Perugia, Italy, Tel.: +39 075 5783989, Fax: +39 075 5716083, E-mail:

Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p<0.05) and increased retained platelets (from 72.3 ± 2.3% to 81.1 ± 1.8%, p<0.05) in the O'Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, p<0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.
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http://dx.doi.org/10.1160/TH15-04-0300DOI Listing
January 2016

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia.

Haematologica 2016 Jan 9;101(1):46-56. Epub 2015 Oct 9.

Department of Medicine, Section of Internal and Cardiovascular Medicine, University of Perugia, Italy

Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin αIIbβ3. The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of αIIbβ3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β3 del647-686 mutation and Chinese hamster ovary cells expressing different αIIbβ3-activating mutations, we showed that reduced surface expression of αIIbβ3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of αIIbβ3-mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β3-transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated αIIbβ3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia.
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http://dx.doi.org/10.3324/haematol.2015.130849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697891PMC
January 2016

Prevalence of hemostatic alterations in patients with recurrent spontaneous subconjunctival hemorrhage.

Clin Chem Lab Med 2016 Jan;54(1):97-103

Background: Subconjunctival hemorrage (SCH) is a frequent, mild bleeding manifestation and a common cause of consultation. Hemostatic alterations are possible causes of SCH but their role and prevalence is unknown. We assessed the prevalence of hemostatic abnormalities in patients with spontaneous, recurrent SCH to clarify the role of the hemostasis laboratory in this clinical setting.

Methods: A total of 105 SCH patients (21-78 years, 65 females) with no identifiable cause (hypertension-trauma-conjunctivitis) or concomitant treatments (NSAIDs- aspirin-oral anticoagulants-antiplatelet agents) and 53 age and sex-matched healthy controls (HCs) (22-72 years, 29 females) were evaluated for skin bleeding time, PFA-100®, blood clotting screening, platelet count, light transmission aggregomery, VWF:Ag, VWF:RCo, RIPA, FVIII activity, FXIII antigen and activity and ISTH Bleeding Severity Score (BSS).

Results: Prevalence of hemostatic abnormalities was not higher in the SCH population than in HCs BSS was 0.83 (95% CI 0.62-1.06) in SCH and 0.66 (0.37-0.95) in HC (p=NS). Type I Von Willebrand disease was diagnosed in one SCH and none HC patients, a prevalence not significantly different (p=NS by χ2).

Conclusions: The prevalence of hemostatic alterations in patients with recurrent, spontaneous SCH is not different from the general population; hemostatic screening or second level tests are of no use in patients with recurrent SCH and no other bleedings.
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http://dx.doi.org/10.1515/cclm-2015-0274DOI Listing
January 2016