Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

Kinetics and regulation of coagulation factor X activation by intrinsic tenase on phospholipid membranes.

BACKGROUND: Factor X activation by the phospholipid-bound intrinsic tenase complex is a critical membrane-dependent reaction of blood coagulation. Its regulation mechanisms are unclear, and a number of questions regarding diffusional limitation, pathways of assembly and substrate delivery remain open. METHODS: We develop and analyze here a detailed mechanism-driven computer model of intrinsic tenase on phospholipid surfaces. Three-dimensional reaction-diffusion-advection and stochastic simulations were used where appropriate. RESULTS: Dynamics of the system was predominantly non-stationary under physiological conditions. In order to describe experimental data, we had to assume both membrane-dependent and solution-dependent delivery of the substrate. The former pathway dominated at low cofactor concentration, while the latter became important at low phospholipid concentration. Factor VIIIa-factor X complex formation was the major pathway of the complex assembly, and the model predicted high affinity for their lipid-dependent interaction. Although the model predicted formation of the diffusion-limited layer of substrate for some conditions, the effects of this limitation on the fXa production were small. Flow accelerated fXa production in a flow reactor model by bringing in fIXa and fVIIIa rather than fX. CONCLUSIONS: This analysis suggests a concept of intrinsic tenase that is non-stationary, employs several pathways of substrate delivery depending on the conditions, and is not particularly limited by diffusion of the substrate.

Force balance ratio is a robust predictor of arterial thrombus stability.

Thrombus formation on a damaged vessel wall can lead to the formation of a stable occlusive/subocclusive clot or unstable embolizing thrombus. Both outcomes can cause significant health damage. The mechanisms that regulate maximum thrombus size, its stability, and embolization in both micro- and macrocirculation are poorly understood. To investigate the impact of flow and intrathrombus forces on the stability of homogeneous and heterogeneous platelet thrombi in a wide range of thrombus geometries, critical interplatelet forces, vessel diameters, and hydrodynamic conditions, we took advantage of the recently developed in silico models. To perform analysis of thrombus stability/embolization in arterioles, we used our previously developed particle-based 2D model with a single-platelet resolution. Its results and predictions were further extended to a 3D case and the large spatial scales of arteries using novel particle-based and continuum 3D models. We found a robust quantitative parameter, termed force balance ratio, which quantifies the balance between destabilizing hydrodynamic and stabilizing interplatelet forces. This parameter predicts whether a homogeneous thrombus (or the shell of a heterogeneous thrombus) with a particular value of critical interplatelet forces will embolize under given hydrodynamic conditions. Our simulations also predict that, for a given magnitude of critical interplatelet forces, the longer thrombi are more stable than the shorter ones. Furthermore, the aggregates formed on top of the severe stenosis are more stable than thrombi formed at moderate stenosis. Taken together, our results give new insights into the interplay between critical interplatelet forces, local hydrodynamics, and overall thrombus stability against the flow.

Chemical Adjustment of Fibrinolysis.

Fibrinolysis is the process of the fibrin-platelet clot dissolution initiated after bleeding has been stopped. It is regulated by a cascade of proteolytic enzymes with plasmin at its core. In pathological cases, the balance of normal clot formation and dissolution is replaced by a too rapid lysis, leading to bleeding, or an insufficient one, leading to an increased thrombotic risk. The only approved therapy for emergency thrombus lysis in ischemic stroke is recombinant tissue plasminogen activator, though streptokinase or urokinase-type plasminogen activators could be used for other conditions. Low molecular weight compounds are of great interest for long-term correction of fibrinolysis dysfunctions. Their areas of application might go beyond the hematology field because the regulation of fibrinolysis could be important in many conditions, such as fibrosis. They enhance or weaken fibrinolysis without significant effects on other components of hemostasis. Here we will describe and discuss the main classes of these substances and their mechanisms of action. We will also explore avenues of research for the development of new drugs, with a focus on the use of computational models in this field.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

The Ways of the Virus: Interactions of Platelets and Red Blood Cells with SARS-CoV-2, and Their Potential Pathophysiological Significance in COVID-19.

The hematological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important in COVID-19 pathophysiology. However, the interactions of SARS-CoV-2 with platelets and red blood cells are still poorly understood. There are conflicting data regarding the mechanisms and significance of these interactions. The aim of this review is to put together available data and discuss hypotheses, the known and suspected effects of the virus on these blood cells, their pathophysiological and diagnostic significance, and the potential role of platelets and red blood cells in the virus's transport, propagation, and clearance by the immune system. We pay particular attention to the mutual activation of platelets, the immune system, the endothelium, and blood coagulation and how this changes with the evolution of SARS-CoV-2. There is now convincing evidence that platelets, along with platelet and erythroid precursors (but not mature erythrocytes), are frequently infected by SARS-CoV-2 and functionally changed. The mechanisms of infection of these cells and their role are not yet entirely clear. Still, the changes in platelets and red blood cells in COVID-19 are significantly associated with disease severity and are likely to have prognostic and pathophysiological significance in the development of thrombotic and pulmonary complications.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

BACKGROUND: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. METHODS: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. RESULTS: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. CONCLUSIONS: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. IMPACT: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Platelets provide robustness of spatial blood coagulation to the variation of initial conditions.

Activated platelets provide phospholipid surface and secrete coagulation factors, enhancing blood clotting. We investigated the role of platelets in the regulation of blood coagulation spatial dynamics. We activated blood clotting with tissue factor-bearing (TF) surface in platelet-rich plasma (PRP) or platelet-free plasma (PFP). When blood coagulation was initiated by high TF density, clot growth rate (V) in PRP (2 × 105/µL platelets) was only 15 % greater than in PFP. Spatial distribution of thrombin in PRP had a peak-like shape in the area of the fibrin clot edge, while in PFP thrombin was distributed in the shape of descending plateau. Platelet inhibition with prostaglandin E1 or cytochalasin D made spatial thrombin distribution look like in the case of PFP. Inhibition of blood coagulation by natural endogenous inhibitor heparin was diminished in PRP, while the effect of the exogenous or artificial inhibitors (rivaroxaban, nitrophorin, hirudin) remained undisturbed in the presence of platelets. Ten times decrease of the TF surface density greatly depressed blood coagulation in PFP. In PRP only clotting initiation phase was, while the propagation phase remained intact. Coagulation factor deficiency greatly reduced amount of thrombin and decreased V in PFP rather than in PPR. Thus, platelets were redundant for clotting in normal plasma under physiological conditions but provided robustness of the coagulation system to the changes in initial conditions.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Integral assays of hemostasis in hospitalized patients with COVID-19 on admission and during heparin thromboprophylaxis.

BACKGROUND: Blood coagulation abnormalities play a major role in COVID-19 pathophysiology. However, the specific details of hypercoagulation and anticoagulation treatment require investigation. The aim of this study was to investigate the status of the coagulation system by means of integral and local clotting assays in COVID-19 patients on admission to the hospital and in hospitalized COVID-19 patients receiving heparin thromboprophylaxis. METHODS: Thrombodynamics (TD), thromboelastography (TEG), and standard clotting assays were performed in 153 COVID-19 patients observed in a hospital setting. All patients receiving treatment, except extracorporeal membrane oxygenation (ECMO) patients (n = 108), were administered therapeutic doses of low molecular weight heparin (LMWH) depending on body weight. The ECMO patients (n = 15) were administered unfractionated heparin (UFH). RESULTS: On admission, the patients (n = 30) had extreme hypercoagulation by all integral assays: TD showed hypercoagulation in ~75% of patients, while TEG showed hypercoagulation in ~50% of patients. The patients receiving treatment showed a significant heparin response based on TD; 77% of measurements were in the hypocoagulation range, 15% were normal, and 8% remained in hypercoagulation. TEG showed less of a response to heparin: 24% of measurements were in the hypocoagulation range, 59% were normal and 17% remained in hypercoagulation. While hypocoagulation is likely due to heparin treatment, remaining in significant hypercoagulation may indicate insufficient anticoagulation for some patients, which is in agreement with our clinical findings. There were 3 study patients with registered thrombosis episodes, and all were outside the target range for TD parameters typical for effective thromboprophylaxis (1 patient was in weak hypocoagulation, atypical for the LMWH dose used, and 2 patients remained in the hypercoagulation range despite therapeutic LMWH doses). CONCLUSION: Patients with COVID-19 have severe hypercoagulation, which persists in some patients receiving anticoagulation treatment, while significant hypocoagulation is observed in others. The data suggest critical issues of hemostasis balance in these patients and indicate the potential importance of integral assays in its control.

New Hybrid Tetrahydropyrrolo[3,2,1-ij]quinolin-1-ylidene-2-thioxothiazolidin-4-ones as New Inhibitors of Factor Xa and Factor XIa: Design, Synthesis, and In Silico and Experimental Evaluation.

Despite extensive research in the field of thrombotic diseases, the prevention of blood clots remains an important area of study. Therefore, the development of new anticoagulant drugs with better therapeutic profiles and fewer side effects to combat thrombus formation is still needed. Herein, we report the synthesis and evaluation of novel pyrroloquinolinedione-based rhodanine derivatives, which were chosen from 24 developed derivatives by docking as potential molecules to inhibit the clotting factors Xa and XIa. For the synthesis of new hybrid derivatives of pyrrolo[3,2,1-ij]quinoline-2-one, we used a convenient structural modification of the tetrahydroquinoline fragment by varying the substituents in positions 2, 4, and 6. In addition, the design of target molecules was achieved by alkylating the amino group of the rhodanine fragment with propargyl bromide or by replacing the rhodanine fragment with 2-thioxoimidazolidin-4-one. The in vitro testing showed that eight derivatives are capable of inhibiting both coagulation factors, two compounds are selective inhibitors of factor Xa, and two compounds are selective inhibitors of factor XIa. Overall, these data indicate the potential anticoagulant activity of these molecules through the inhibition of the coagulation factors Xa and XIa.

Accelerated death of megakaryocytes from Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients.

Different modeling approaches in the simulation of extrinsic coagulation factor X activation: Limitations and areas of applicability.

Proteolytic reactions on the phospholipid membrane surface, so-called "membrane-dependent" reactions, play central role in the process of blood clotting. One particularly important example is FX activation by the extrinsic tenase (VIIa/TF). Here we constructed three mathematical models of FX activation by VIIa/TF: (A) a homogeneous "well-mixed" model, (B) a two-compartment "well-mixed" model, (C) a heterogeneous model with diffusion, to investigate the impact and importance of inclusion of each complexity level. All models provided good description of the reported experimental data and were equivalently applicable for <40 µM of phospholipids. Model C provided better predictions than A, B in the presence of TF-negative phospholipid microparticles. Models predicted that for high TF surface density (STF ) and FX deficiency the FX activation rate was limited by the rate of FX binding to the membrane. For low STF and excess of FX the reaction rate was limited by the tenase formation rate. The analysis of the substrate delivery pathways revealed that FX bound to VIIa/TF predominantly from solution for STF >2.8 × 10-3  nmol/cm2 and from the membrane for lower STF . We proposed the experimental setting to distinguish between the collision-limited and non-collision-limited binding. The analysis of models in flow and non-flow conditions revealed that the model of a vesicle in flow might be substituted by model C in the absence of the substrate depletion. Together, this study was the first which provided the direct comparison of more simple and more complex models. The reaction mechanisms were studied in a wide range of conditions.

Platelet Hemostasis Reactions at Different Temperatures Correlate with Intracellular Calcium Concentration.

Hypo- and hyperthermia affect both primary and secondary hemostasis; however, there are controversial data concerning platelet activation and the underlying mechanisms under hypo- and hyperthermia. The discrepancies in the data could be partly explained by different approaches to hemostatic reactions analysis. We applied a new LaSca-TMF laser particle analyzer for a simultaneous fluorescence and laser scattering analysis of platelet responses at different temperatures. Human platelets were activated by ADP in a wide range of temperatures, and platelet transformations (e.g., a shape change reaction, aggregation and clot formation) and the intracellular calcium concentration ([Ca2+]i) were analyzed by LaSca-TMF and confocal microscopy. The platelet shape change reaction gradually increased with a rising temperature. The platelet aggregation strongly decreased at low ADP concentrations with the augmentation of the temperature and was independent of the temperature at high ADP concentrations. In contrast, the clotting time decreased with a temperature increase. Similar to the aggregation response, a rise in [Ca2+]i triggered by low ADP concentrations was higher under hypothermic conditions and the differences were independent of the temperature at high ADP concentrations. We showed that the key reactions of cellular hemostasis are differentially regulated by temperature and demonstrated for the first time that an accelerated aggregation under hypothermic conditions directly correlated with an increased level in [Ca2+]i in platelets.

Procoagulant Activity in Amniotic Fluid Is Associated with Fetal-Derived Extracellular Vesicles.

Procoagulant activity in amniotic fluid (AF) is positively correlated with phosphatidylserine (PS) and tissue factor (TF)-expressing(+) extracellular vesicles (EVs). However, it is unknown if pathological fetal conditions may affect the composition, phenotype, and procoagulant potency of EVs in AF. We sought to evaluate EV-dependent procoagulant activity in AF from pregnant people with fetuses with or without diagnosed chromosomal mutations. AF samples were collected by transabdominal amniocentesis and assessed for common karyotype defects (total n = 11, 7 healthy and 4 abnormal karyotypes). The procoagulant activity of AF was tested using a fibrin generation assay with normal pooled plasma and plasmas deficient in factors XII, XI, IX, X, V, and VII. EV number and phenotype were determined by flow cytometry with anti-CD24 and anti-TF antibodies. We report that factor-VII-, X-, or V-deficient plasmas did not form fibrin clots in the presence of AF. Clotting time was significantly attenuated in AF samples with chromosomal mutations. In addition, CD24+, TF+, and CD24+ TF+ EV counts were significantly lower in this group. Finally, we found a significant correlation between EV counts and the clotting time induced by AF. In conclusion, we show that AF samples with chromosomal mutations had fewer fetal-derived CD24-bearing and TF-bearing EVs, which resulted in diminished procoagulant potency. This suggests that fetal-derived EVs are the predominant source of procoagulant activity in AF.

Traumatic vessel injuries initiating hemostasis generate high shear conditions.

Blood flow is a major regulator of hemostasis and arterial thrombosis. The current view is that low and intermediate flows occur in intact healthy vessels, whereas high shear levels (>2000 s-1) are reached in stenosed arteries, notably during thrombosis. To date, the shear rates occurring at the edge of a lesion in an otherwise healthy vessel are nevertheless unknown. The aim of this work was to measure the shear rates prevailing in wounds in a context relevant to hemostasis. Three models of vessel puncture and transection were developed and characterized for a study that was implemented in mice and humans. Doppler probe measurements supplemented by a computational model revealed that shear rates at the edge of a wound reached high values, with medians of 22 000 s-1, 25 000 s-1, and 7000 s-1 after puncture of the murine carotid artery, aorta, or saphenous vein, respectively. Similar shear levels were observed after transection of the mouse spermatic artery. These results were confirmed in a human venous puncture model, where shear rates in a catheter implanted in the cubital vein reached 2000 to 27 000 s-1. In all models, the high shear conditions were accompanied by elevated levels of elongational flow exceeding 1000 s-1. In the puncture model, the shear rates decreased steeply with increasing injury size. This phenomenon could be explained by the low hydrodynamic resistance of the injuries as compared with that of the downstream vessel network. These findings show that high shear rates (>3000 s-1) are relevant to hemostasis and not exclusive to arterial thrombosis.

Ex vivo observation of granulocyte activity during thrombus formation.

BACKGROUND: The process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors. RESULTS: Thrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s-1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90-120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients' platelets were replaced with healthy donor's platelets the granulocytes' crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes' behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers. CONCLUSIONS: We have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

Longitudinal multiparametric characterization of platelet dysfunction in COVID-19: Effects of disease severity, anticoagulation therapy and inflammatory status.

INTRODUCTION: Defects of platelet functional responses in COVID-19 were reported, but their origin and pathophysiological significance are unclear. The objective of this study was to characterize the thrombocytopathy in COVID-19. MATERIALS AND METHODS: Analysis of platelet functional responses to activation by flow cytometry and aggregometry in 46 patients with confirmed COVID-19 of different severity (non-ICU, ICU, and ECMO) over the course of hospitalization alongside with plasma coagulation, inflammatory markers (CRP, fibrinogen, NETosis assays in smears) was performed. RESULTS AND CONCLUSIONS: All patients had increased baseline percentage of procoagulant platelets (healthy: 0.9 ± 0.5%; COVID-19: 1.7 ± 0.6%). Patients had decreased agonist-induced platelet GPIb shedding (1.8 ± 0.7 vs 1.25 ± 0.4), P-Selectin exposure (1.51 ± 0.21 vs 1.1 ± 0.3) and aggregation. The values of these parameters among the non-ICU and ICU cohorts differed modestly, while the ECMO cohort differed significantly. Only ECMO patients had pronounced thrombocytopenia. While inflammatory markers improved over time, the observed platelet functional responses changed only moderately. SARS-CoV-2 RNA was found in 8% of blood samples and it did not correlate with platelet counts or responses. All patients had increased NETosis that moderately correlated with platelet dysfunction. High cumulative dosages of LMWH (average > 12,000 IU/day over 5 days) resulted in an improvement in platelet parameters. The observed pattern of platelet refractoriness was reproduced by in vitro pre-treatment of washed platelets with subnanomolar thrombin or perfusion of blood through a collagen-covered flow chamber. We conclude that platelet dysfunction in COVID-19 is consistent with the intravascular-coagulation-induced refractoriness rather than with an inflammation-induced mechanism or a direct activation by the virus.

Analysis of microvascular thrombus mechanobiology with a novel particle-based model.

Platelet accumulation at the site of a vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood. Here we describe a novel computational model of microvascular clot formation for the detailed analysis of thrombus mechanics. We adopt a previously developed two-dimensional particle-based model focused on the thrombus shell formation and revise it to introduce the platelet agonists. Blood flow is simulated via a computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process. The new model qualitatively reproduces the enhanced thrombus formation due to dense granule secretion, in line with in vivo findings, and provides a mechanism for the thrombin confinement at the early stages of clot formation. Our calculations also predict that the release of platelet dense granules results in the additional mechanical stabilization of the inner layers of thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of a thrombus, which are expected to result in the mechanical disruptions at the later stages of clot formation.