Intravenous enoxaparin guided by anti-Xa in venovenous extracorporeal membrane oxygenation: A retrospective, single-center study.

BACKGROUND: Unfractionated heparin is used as the most common anticoagulation for venovenous extracorporeal membrane oxygenation (VV ECMO) patients. However, it is accompanied by frequent bleeding and thrombotic complications. The aim of the study was to demonstrate the feasibility of Enoxaparin anticoagulation for VV ECMO patients. METHODS: This study is a retrospective analysis of VV ECMO patients on continuous intravenous Enoxaparin anticoagulation. The primary outcome was the incidence of bleeding, thrombotic, and neurological complications during ECMO support. The secondary outcome was an analysis of secondary and primary hemostasis profiles. RESULTS: Data from 38 patients were analyzed in this study. The incidence of bleeding complications was 5.3%, for thrombotic complications it was 2.6% and for neurological (bleeding/ischemic events) complications it was 10.5%. The targeted anti-Xa activity of 0.4-0.6 IU/mL was achieved and maintained during whole ECMO period in 28 patients (73.8%), not affecting the hemocoagulation profile represented by APTT-r 1.15 ± 0.2, TT 18.67 ± 3.35 s, PT/INR 1.21 ± 0.19, fibrinogen 5.39 ± 1.49 g/L, antithrombin, and platelet count. Primary hemostasis pathology was diagnosed in all patients by PFA 200 tests Col/EPI 279 ± 38 s and Col/ADP 249 ± 66 s. The running time of ECMO was 7.8 ± 3.4 days. CONCLUSIONS: Enoxaparin anticoagulation appears to be feasible for VV ECMO patients without an increase in adverse events. Further larger-sampled and comparative studies are needed in the future to support our findings.

A novel approach to laboratory assessment and reporting of platelet von Willebrand factor.

The interaction of platelets with von Willebrand factor is essential for primary hemostasis. Concentration and activity of plasma von Willebrand factor are routine parameters in the assessment of hemostasis disorders. In addition to plasma von Willebrand factor, platelet von Willebrand factor, synthesized in megakaryocytes and stored in α-granules of circulating platelets, is known to contribute to primary hemostasis and the microenvironment of thrombus formation. The laboratory assessment of platelet von Willebrand factor however is cumbersome and not widely established as a routine parameter. We here propose a method for laboratory assessment and reporting of platelet von Willebrand factor potentially useful for laboratory routines in specialized laboratories. Our model allows to describe platelet von Willebrand factor as 1. the concentration of platelet von Willebrand factor in whole blood, 2. the amount of platelet von Willebrand factor in a sample with a defined concentration of 1000 platelets/nl, and 3. the concentration of platelet von Willebrand factor in one platelet. According to our results in healthy individuals, the proportion of platelet von Willebrand factor activity is estimated to be about 10% of total von Willebrand factor in human plasma under physiological circumstances. The concentration of platelet von Willebrand factor is estimated to be 0.4 IU/ml in a sample with a defined concentration of 1000 platelets/nl and to be about 42 IU/ml in one platelet (both expressed as VWF:Ag).

Extracorporeal membrane oxygenation seems to induce impairment of primary hemostasis pathology as measured by a Multiplate analyzer: An observational retrospective study.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) support is often associated with bleeding complications caused by secondary or primary hemostasis pathology. However, there are limited data investigating primary hemostasis using Multiplate aggregometry with specific diagnostics tests for vWF (von Willebrand factor) deficiency. AIMS: The aim of this study was to find out whether short-term ECMO produces the pathology of primary hemostasis that is detected by Multiplate aggregometry and to investigate the pathology of vWF. METHODS: In this study, blood samples of 20 patients undergoing lung transplantations with short-term perioperative ECMO support were analyzed. The multimeric structure, the levels of von Willebrand factor antigen (vWF), ristocetin cofactor (RCo), collagen-binding protein (CB), and the results of multiple electrode aggregometry RISTO (ristocetin), ADP (adenosine diphosphate), ASPI (Aspirin®; arachidonic acid), and TRAP (thrombin receptor activating peptide) tests were compared to the samples obtained before and after ECMO support. RESULTS: The Multiplate ADP and RISTO tests showed the presence of significant pathology in primary hemostasis after surgery (p < 0.05), suggesting the presence of acquired platelet dysfunction. Although the RISTO tests suggest the presence of acquired vWF deficiency, laboratory tests for vWF antigen and RCo and CB tests showed an increase in this case. The multimeric structure of vWF did not show clinically significant deterioration. CONCLUSIONS: Multiple aggregometry ADP, ASPI, and TRAP tests seem to be able to detect primary hemostasis pathology (platelets aggregation and adhesion pathology) that is present during short-term perioperative ECMO support in lung transplantation procedures. Interestingly, RISTO tests seem to be more suitable for the diagnosis of platelet dysfunction than the diagnosis of acquired vWF deficiency in this situation.

Diagnosing Czech Patients with Inherited Platelet Disorders.

A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.

[How I explore hémostatic abnormalities in pediatrics]. / Comment j'explore. Une anomalie du bilan d'hémostase en pédiatrie.

Hemostasis work-up is frequently requested in pediatric cares and can often seem complicated to interpret when certain results return to be abnormal. In addition, these biological tests are very sensitive and several pre-analytical conditions can influence them and skew the results, leading to erroneous analyzes and diagnoses. Indeed, systemic inflammation, anemia or even only the delay between blood sampling and analysis can make the results more difficult to be interpreted. However, when the tests have been carried out under good conditions, having in mind a few basic knowledge of hemostasis can easily help to first distinguish a pathology of primary hemostasis from a coagulopathy. Secondly, depending on the abnormal biological tests, complementary oriented assays may then be requested, ideally in a laboratory specialized in hemostasis, in order to confirm or rule out a true hemorrhagic pathology.

Le bilan d'hémostase standard, fréquemment demandé en pédiatrie, peut souvent sembler compliqué à interpréter lorsque certains résultats reviennent anormaux. De plus, l'influence des conditions pré-analytiques, parfois méconnues, sur ces tests biologiques extrêmement sensibles peut en fausser les résultats et entraîner des analyses et des diagnostics erronés. En effet, des paramètres tels que l'inflammation, un contexte d'anémie ou encore un délai trop important entre le prélèvement sanguin et l'analyse peuvent rendre les résultats ininterprétables. Lorsque les tests ont été réalisés dans de bonnes conditions, quelques bases de physiologie de l'hémostase ainsi que quelques spécificités liées à la pédiatrie permettent facilement de s'orienter vers une pathologie de l'hémostase primaire ou de la coagulation. Dans un second temps, en fonction des tests biologiques altérés, des dosages complémentaires orientés peuvent être demandés, idéalement dans un laboratoire spécialisé en hémostase, afin d'affirmer ou infirmer une véritable pathologie hémorragique.

Primary Hemostasis in Chronic Liver Disease and Cirrhosis: What Did We Learn over the Past Decade?

Changes in primary hemostasis have been described in patients with chronic liver disease (CLD) and cirrhosis and are still subject to ongoing debate. Thrombocytopenia is common and multifactorial. Numerous studies also reported platelet dysfunction. In spite of these changes, primary hemostasis seems to be balanced. Patients with CLD and cirrhosis can suffer from both hemorrhagic and thrombotic complications. Variceal bleeding is the major hemorrhagic complication and is mainly determined by high portal pressure. Non portal hypertension-related bleeding due to hemostatic failure is uncommon. Thrombocytopenia can complicate management of invasive procedures in CLD patients. Recently, oral thrombopoietin agonists have been approved to raise platelets before invasive procedures. In this review we aim to bundle literature, published over the past decade, discussing primary hemostasis in CLD and cirrhosis including (1) platelet count and the role of thrombopoietin (TPO) agonists, (2) platelet function tests and markers of platelet activation, (3) von Willebrand factor and (4) global hemostasis tests.

Dasatinib inhibits coated-platelet generation in patients with chronic myeloid leukemia.

Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.

Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders.

Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p = 0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.

[A diagnostic approach to relatively common mild bleeding symptoms: screening of congenital mild bleeding disorders].

Although mild skin and mucosal bleeding symptoms are common in patients with inherited bleeding disorders, both can also be observed in healthy individuals. These bleeding symptoms may be caused by disorders of primary hemostasis (i.e., von Willebrand disease and platelet function disorders), mild deficiencies of clotting factors and some rare hyper fibrinolytic disorders are also included. To date, mild bleeding symptoms pose a frequent and substantial diagnostic challenge, and the definitive diagnosis of inherited bleeding disorders is not feasible in a vast majority of individuals despite an extensive laboratory workup. However, it must be diagnosed precisely to avoid severe complications and decrease bleeding risks before invasive procedures. Thus, a comprehensive diagnostic approach, from structured patient history to highly specialized laboratory diagnosis, is warranted to establish diagnosis of inherited bleeding disorders. Furthermore, the prophylaxis should be offered to prevent excessive bleeding in these patients with bleeding symptoms, even if they remain undiagnose of bleeding disorders.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Comprehensive investigation of platelet function in patients with cirrhosis.

Cirrhosis presents with thrombocytopenia and possibly thrombocytopathy. Previous studies exploring platelet function gave conflicting results and most controversies are explained by the variety of methods employed for investigation. We sought to assess in-vitro the overall platelet function in cirrhosis. We investigated 34 patients by using the following tests. (i)Aggregometry. (ii)Measurement of the content of platelet granules. (iii)Cytometric platelet activation. (iv)Plasmatic markers of in-vivo platelet activation. (v)Platelet procoagulant activity by thrombin generation (TG) in platelet-rich plasma (PRP). TG measured in PRP for patients and controls was similar. Platelets from patients with cirrhosis showed reduction of aggregation and secretion of ATP. Similar results were observed for platelet activation parameters such as P-selectin expression and PAC-1 platelet binding. Plasma levels of ßeta-thromboglobulin and soluble P-selectin, were increased in patients-vs-controls. In contrast, there were no patients-vs-controls differences for plasmatic platelet-factor-4. Results are consistent with a state of in-vivo platelet activation and decreased in-vitro aggregation. Since bleeding events following invasive procedures are uncommon in cirrhosis, we speculate that in-vitro aggregometry testing does not reflect the situation occurring in-vivo. Results of the study and pathophysiological considerations support the conclusion that platelet function in cirrhosis as determined by aggregometry, although somewhat impaired, may support the overall hemostatic potential, which is needed for most invasive interventions. These conclusions are in line with the recommendations of international guidelines, warning against indiscriminate use of prophylactic preprocedural administration of platelets before invasive procedures. Decision on platelet support should not be made based on in-vitro laboratory testing for platelet function.

ECMO produces very rapid changes in primary hemostasis detected by PFA-200 during lung transplantation: An observational study.

BACKGROUND: The pathology of primary hemostasis is a common complication of extracorporeal membrane oxygenation (ECMO) support. Scientific data describing its changes in patients on short-term ECMO support and the ability and speed of the restoration of its functions are limited. AIMS: The aim of this study was to describe the pathology of primary hemostasis induced by short-term ECMO support and its development over time using PFA-200, ROTEM platelet, and von Willebrand factor (vWF) analyses. METHODS: In patients undergoing lung transplantation surgery using intraoperative veno-arterial ECMO support, blood samples were analyzed using the following tests: PFA-200, ROTEM platelet tests, vWF antigen, ristocetin cofactor (RCo), and collagen-binding protein (CB) before, during, and after ECMO support. RESULTS: Blood samples from 32 patients were analyzed. All 3 PFA-200 tests (COL/EPI, COL/ADP, and COL/P2Y) showed significant deterioration during ECMO support with rapid restoration after ECMO cessation (p < 0.05), suggesting an ECMO-induced primary hemostasis disorder. A significant increase of vWF antigen after ECMO cessation (p < 0.05) was found with an increase of RCo and CB levels, although it was not significant (p > 0.05). CONCLUSIONS: Short-term ECMO support induces primary hemostasis pathology. It occurs immediately after initiation but is rapidly restored after ECMO cessation, which is detectable by PFA-200. Despite there being persistent platelet dysfunction after ECMO cessation, as seen with the ROTEM platelet results, the increased levels of vWF antigen might explain the normal results of primary hemostasis detected by PFA-200.

Primary Hemostasis Disorders as a Cause of Heavy Menstrual Bleeding in Women of Reproductive Age.

Heavy menstrual bleeding (HMB) is a common clinical condition affecting adolescent and adult women and compromising their quality of life. Primary hemostasis disorders, affecting platelet plug formation, can be the underlying cause of HMB. They comprise a heterogeneous group of diseases with Von Willebrand disease (VWD) being the most commonly diagnosed; other disorders in this group that have been linked to HMB include (a) Glanzmann thrombasthenia, (b) Bernard-Soulier syndrome, (c) Hermansky-Pudlak syndrome, (d) immune thrombocytopenia (ITP), and (e) Ehlers-Danlos syndromes (EDS) and hypermobility spectrum disorders (HSD). Diagnosing these diseases can be challenging, as the basic laboratory investigations can be within the normal range. Thus, identification of specific clinical features and a thorough hematologic workup can be very important, providing the correct diagnosis. Proper diagnosis of the underlying disorder is important, as management may vary accordingly. Although disease-specific management guidelines exist for some of these disorders such as VWD and ITP, due to the rarity of most primary hemostasis disorders, the best approach for the management of HMB in these women remains elusive. The goal of this study was to create an informative, comprehensive review of the primary hemostasis disorders that have been linked to HMB. This study provides a summary of the basic published information regarding epidemiology, pathophysiology, clinical phenotype, diagnosis, and treatment of HMB in those diseases and serves as a reference guide for further reading.

Combination of Primary Hemostatic Disorders and Atrial Fibrillation Increases Bleeding Events Following Transcatheter Aortic Valve Replacement.

Background Patients with atrial fibrillation (AF) are likely to have a poor prognosis including bleedings following transcatheter aortic valve replacement (TAVR). Closure time of adenosine diphosphate (CT-ADP) is a primary hemostasis point-of-care test and is a predictor of bleeding events following TAVR. We aimed to evaluate the impact of ongoing primary hemostatic disorders on bleeding events in TAVR patients with AF. Methods We enrolled 878 patients from our prospective registry. The primary endpoint was VARC-2 major/life-threatening bleeding complications (MLBCs) at 1 year after TAVR and secondary endpoint was major adverse cardiac and cerebrovascular events (MACCEs) at 1 year, defined as a composite of all-cause death, myocardial infarction, stroke, and heart failure hospitalization. Ongoing primary hemostatic disorder was defined by a postprocedural CT-ADP >180 seconds. Results Patients with AF had a higher incidence of MLBCs (20 vs. 12%, p = 0.002), MACCE (29 vs. 20%, p = 0.002), and all-cause mortality (15 vs. 8%, p = 0.002) within 1 year compared to non-AF patients. When the cohort was split into four subgroups according to AF and CT-ADP >180 seconds, patients with AF and CT-ADP >180 seconds had the highest risk of MLBCs and MACCE. Multivariate Cox regression analysis confirmed that the patients with AF and CT-ADP >180 seconds had 3.9-fold higher risk of MLBCs, whereas those patients were no longer associated with MACCE after the adjustment. Conclusion In TAVR patients, AF with postprocedural CT-ADP >180 seconds was strongly associated with MLBCs following TAVR. Our study suggests that persistent primary hemostatic disorders contribute to a higher risk of bleeding events particularly in AF patients.

Delta Storage Pool Deficiency: A Pediatric Case Report and Review of the Literature.

Platelet storage deficiencies are a heterogeneous group of bleeding disorders of variable severity caused by decreased number or content of platelet granules. We present the case of a 10-year-old patient with no personal history of previous bleeding who was admitted to the emergency department due to menorrhagia and mucocutaneous pallor. Common disorders of primary and secondary hemostasis were ruled out. Subsequently, a study of electron microscopy of platelets was performed, which reported the presence of alpha granules with a decreased number of dense granules. Currently, the patient receives treatment with tranexamic acid during menstrual periods, supplementation with ferrous sulfate, and oral contraceptives, achieving control of bleeding episodes.

Whole blood platelet impedance aggregometry with the ROTEM platelet device: comparison of 2 anticoagulants and storage times for the establishment of canine reference intervals.

The ROTEM platelet device, a point-of-care whole blood platelet impedance aggregometer, is an add-on to the rotational thromboelastometry ROTEM delta device. The latter has been validated in dogs. We examined whether canine whole blood is suited for analysis with the ROTEM platelet device using adenosine-5'-diphosphate (ADP) and arachidonic acid (ARA) as agonists for platelet activation, and if there are significant differences between sample storage times and anticoagulants used. Subsequently, we determined canine reference intervals (RIs) for the ROTEM platelet device for ADP and ARA. In a pilot study, we examined whole blood from 7 dogs after 15-min and 60-min storage of lithium-heparinized samples and 40-min and 80-min storage of hirudinized samples. Statistical analysis showed no significant differences between ROTEM platelet device results for both ADP and ARA in lithium-heparin and hirudin anticoagulated canine whole blood. Lithium-heparinized blood samples analyzed after 15-min storage had the lowest coefficient of variation. RIs were determined for heparinized whole blood samples from 49 dogs after 15 min of storage.

Gestational Diabetes Melitus and Cord Blood Platelet Function Studied via the PFA-100 System.

Neonatal platelet hemostasis, although it has been well described over the recent years, remains elusive in specific patient populations, including neonates from high-risk pregnancies, such as those complicated with gestational diabetes mellitus (GDM). We aimed at evaluating the platelet function of neonates born to mothers with GDM using the platelet function analyzer (PFA-100). Cord blood samples were drawn from each subject and tested with two different agonists to provide two closure time (CT) values (collagen with epinephrine (COL/EPI) and collagen with adenosine diphosphate (COL/ADP)). A total of 84 and 118 neonates formed the GDM and the control group (neonates from uncomplicated pregnancies), respectively. COL/EPI CTs were prolonged in neonates from the GDM group compared to neonates from the control group, while no statistically significant difference of COL/ADP CTs was noted between the two groups, GDM and the control. Higher COL/ADP CTs were demonstrated in neonates born via cesarean section and in neonates with blood group O. A negative correlation between COL/ADP CT and gestational age, white blood cells (WBCs) and von Willebrand factor (VWF) activity was noted in neonates from the GDM group. In conclusion, neonates from the GDM group demonstrate a more hyporesponsive phenotype of their platelets, in comparison to the control neonates.

Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors.

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway. One Sentence Summary: Platelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

Platelet activation in critically ill COVID-19 patients.

BACKGROUND: Microvascular, arterial and venous thrombotic events have been largely described during severe coronavirus disease 19 (COVID-19). However, mechanisms underlying hemostasis dysregulation remain unclear. METHODS: We explored two independent cross-sectional cohorts to identify soluble markers and gene-expression signatures that discriminated COVID-19 severity and outcomes. RESULTS: We found that elevated soluble (s)P-selectin at admission was associated with disease severity. Elevated sP-selectin was predictive of intubation and death (ROC AUC = 0.67, p = 0.028 and AUC = 0.74, p = 0.0047, respectively). An optimal cutoff value was predictive of intubation with 66% negative predictive value (NPV) and 61% positive predictive value (PPV), and of death with 90% NPV and 55% PPV. An unbiased gene set enrichment analysis revealed that critically ill patients had increased expression of genes related to platelet activation. Hierarchical clustering identified ITG2AB, GP1BB, PPBP and SELPLG to be upregulated in a grade-dependent manner. ROC curve analysis for the prediction of intubation was significant for SELPLG and PPBP (AUC = 0.8, p = 0.046 for both). An optimal cutoff value for PBPP was predictive of intubation with 100% NPV and 45% PPV, and for SELPLG with 100% NPV and 50% PPV. CONCLUSION: We provide evidence that platelets contribute to COVID-19 severity. Plasma sP-selectin level was associated with severity and in-hospital mortality. Transcriptional analysis identified PPBP/CXCL7 and SELPLG as biomarkers for intubation. These findings provide additional evidence for platelet activation in driving critical COVID-19. Specific studies evaluating the performance of these biomarkers are required.

Modulation of platelet functions by Careya sphaerica Roxb. leave extracts.

Platelets form a plug to prevent blood loss and contribute to wound healing. Kradonbok, Careya sphaerica Roxb., is a Thai plant with medicinal properties. Conventionally, leaves of C. sphaerica are being used to help wound healing in Thailand. The present study was aimed to investigate the effect of C. sphaerica on the function of platelet. Four different extracts of leaves of C. sphaerica (distilled water, methanol, ethanol, and chloroform extracts) were prepared. The extracts at 5.0 mg/ml per dose were tested for the effect of C. sphaerica on platelet adhesion and aggregation properties, by employing a microtiter plate approach. The phytochemical identification was done by using gas chromatography-mass spectrometry (GC-MS). Our data revealed that chloroform extract significantly activated thrombin-induced platelet adhesion (105.27 ± 0.11%, P < 0.05). None of the extracts exhibited an improvement in platelet aggregation. Further GC-MS analysis of the chloroform extract revealed five key phytochemical constituents with potential platelet activation properties. In conclusion, our study evaluated platelet activation and potentially wound healing property of C. sphaerica. GC-MS analysis identified potential bioactive phytochemical compounds in C. sphaerica which warrant further investigation to characterize these compounds.