Platelet-derived Microparticles Research Articles

Improving platelet function following prophylactic platelet transfusion in patients with hematological malignancies.

Platelet transfusion is a standard treatment to prevent bleeding in patients with hematological malignancies. Although transfusions can improve platelet count, their impact on platelet function remains controversial. We conducted flow cytometry to assess platelet function before and after transfusion and performed subgroup analyses to examine differences based on blood type, corrected count increment (CCI), and platelet microparticles. Overall, 50 patients who received prophylactic platelet transfusion were enrolled. CD42b expression increased, whereas CD41 expression decreased after transfusion. Apheresis platelets exhibited the lowest expression of PAC-1 and P-selectin when exposed to agonist stimulations. PAC-1 expression increased under high adenosine diphosphate (ADP) stimulation, while P-selectin expression increased under both high ADP and thrombin receptor-activating peptide stimulation. In the subgroup analysis, patients with a CCI >4500 and those with the same blood types exhibited a more significant increase in PAC-1 and P-selectin expression under agonist stimulation. When comparing apheresis platelets collected on different days, only the percentage of platelet-derived microparticles showed a significantincrease. Prophylactic transfusion improved platelet function. Platelet function significantly improved in patients with a CCI >4500, those with the same blood types as that of apheresis platelets, or those with platelet-derived microparticle levels <4.7%. No significant improvement in platelet function was noted after the transfusion of different blood types with acceptable compatibility or the transfusion of incompatible blood types. Our results suggest that transfusing platelets with the same blood type remains the optimal choice.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Platelet-derived microparticles adoptively transfer integrin β3 to promote antitumor effect of tumor-infiltrating T cells

ABSTRACT Approximately two-thirds of hepatocellular carcinoma (HCC) is considered a “cold tumor” characterized by few tumor-infiltrating T cells and an abundance of immunosuppressive cells. Cilengitide, an integrin αvβ3 inhibitor, has failed in clinical trials as a potential anticancer drug. This failure implies that integrin αvβ3 may play an important role in immune cells. However, the expression and potential role of integrin αvβ3 in T cells of HCC patients remain unknown. Here, we established two HCC models and found that cilengitide had a dual effect on the HCC microenvironment by exerting both antitumor effect and immunosuppressive effect on T cells. This may partly explain the failure of cilengitide in clinical trials. In clinical specimens, HCC-infiltrating T cells exhibited deficient expression and activation of integrin β3, which was associated with poor T-cell infiltration into tumors. Additionally, integrin β3 functioned as a positive immunomodulatory molecule to facilitate T-cell infiltration and T helper 1-type immune response in vitro. Furthermore, T cells and platelet-derived microparticles (PMPs) co-culture assay revealed that PMPs adoptively transferred integrin β3 to T cells and positively regulated T cell immune response. This process was mediated by clathrin-dependent endocytosis and macropinocytosis. Our data demonstrate that integrin β3 deficiency on HCC-infiltrating T cells may be involved in shaping the immunosuppressive tumor microenvironment. PMPs transfer integrin β3 to T cells and positively regulate T cell immune response, which may provide a new insight into immune therapy of HCC.

Comparative analysis of the quality of platelet concentrates produced by apheresis procedures, platelet rich plasma, and buffy coat.

Platelet concentrates (PCs) could be prepared using either whole-blood processes or apheresis instruments. During collection, processing and storage, some biochemical and functional changes occur, which may result in quality reduction. Quality evaluation of PCs may be helpful for the precise control of platelet (PLT) inventory to reduce the risk of refractoriness and adverse effects caused by platelet transfusion. The study was aimed to evaluate the quality of PCs which were produced by five processes: apheresis (AP) procedures (using three different cell separators: Amicus, Trima Accel and MCS+ instruments), platelet rich plasma (PRP), and buffy coat (BC). A total of 100 PCs (20 of each group) were assessed in respect of routine quality control, morphology, size distribution, destroyed and activated platelets, and production of platelet-derived microparticles (PMPs). All PCs have satisfied the recommended quality of volume, platelet count, residual WBC count, residual RBC count, pH, and sterility according to the Chinese Technical Manual. There was no difference among the 5 groups in morphology and size of PLT and PMPs. Dynamic light scattering test showed that apheresis PCs showed peaks around 10-20 nm, but not whole blood-derived PCs. PCs prepared by Amicus had the relatively high percentage of destroyed platelet, activated platelets and PMPs than other groups. The data suggested high heterogeneity of PMPs, destroyed and activated platelets in PCs produced by different processes, which might be helpful to manage the platelet inventory for targeted use.

Platelet Exosome-Derived miR-223-3p Regulates Pyroptosis in the Cell Model of Sepsis-Induced Acute Renal Injury by Targeting Mediates NLRP3.

The present study investigated the roles and mechanisms of platelet-derived exosomes in sepsis-induced acute renal injury. The blood samples of septic patients and healthy controls were collected for clinical examination. The plasma levels of miR-223-3p and NLRP3 mRNA were analyzed by qRT-PCR and the serum IL-1β and creatinine levels were quantified by enzyme-linked immunosorbent assay (ELISA). C57BL/6 mice injected with LPS (lipopolysaccharide) were employed as the animal model for sepsis-induced acute renal injury. Human coronary artery endothelial cells (HCAECs) were treated with TNF-α as a cellular model for sepsis-induced endothelial damages. The number of PMP (platelet-derived microparticles) in patients with sepsis was increased. The level of miR-223-3p in the platelet exosomes isolated from the serum sample in patients with sepsis was significantly lower than that of the healthy controls. The level of miR-223-3p was also decreased in the platelet exosomes of mouse model with sepsis-induced acute renal injury. Downregulating miR-223-3p promoted sepsis-induced acute renal injury in mice model, while the administration of miR-223-3p reduced the inflammation in endothelial cells of sepsis-induced acute renal injury. NLRP3 (NLR Family Pyrin Domain Containing 3) was identified as one target of miR-223-3p in the platelet exosomes of sepsis-induced acute kidney injury. miR-223-3p attenuated NLRP3-induced pyroptosis in endothelial cell model of sepsis-induced acute kidney injury. Our data suggest that platelet exosome-derived miR-223-3p negatively regulates NLRP3-dependent inflammasome to suppress pyroptosis in endothelial cells. Decreased miR-223-3p expression promotes the inflammation in sepsis-induced acute renal injury. Targeting miR-223-3p may be developed into a therapeutic approach for sepsis-induced acute renal injury.

Platelets: Physiology and Biochemistry

AbstractThis manuscript represents a republication of a manuscript originally published in STH in 2005. This republication is to help celebrate 50 years of publishing for STH. The original abstract follows.Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross-talk to other blood and vascular cells. Stimulated “sticky” platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet–fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.

Mitochondria Transfer by Platelet-Derived Microparticles Regulates Breast Cancer Bioenergetic States and Malignant Features.

We show that the transfer of foreign mitochondria by microparticles modulates recipient cancer cell metabolic plasticity, leading to greater malignant processes.

Gelatin In Situ Zymography to Study Gelatinase Activity in Colon Cancer Cells Treated with Platelet Microparticles (PMPs).

The platelet-derived microparticles (PMPs) have been connected with tumor progression and metastatic dissemination. PMPs infiltrate solid tumors and transfer platelet-derived cargo to cancer cells. The functional roles of PMPs in cancer progression are still poorly understood. PMPs, incorporated by colorectal cancer (CRC) cells, were shown to upregulate the expression and activity of matrix metalloproteases (MMPs). To investigate the impact of PMPs on the invasive potential of CRC, we established the protocol of dequenched gelatin(DG), fluorescein conjugate assay. The procedure confirms the activity of two gelatinases, namely, MMP2 and MMP9, that digest denatured collagen (gelatin). This "step-by-step" protocol, with notes and comments implemented to human CRC lines with different phenotypes and migratory potentials, should be sufficient to obtain representative and elegant results.

The Comparison of Platelet-Derived Microparticles between Ambulatory Cancer Patients and Controls

Background: Elevated levels of microparticles in cancer have been associated with hypercoagulability; however, the specific subtypes of microparticles are still unclear. We hypothesize that active cancer may increase the level of platelet-derived microparticles from platelet activation and play a role in cancer-associated thrombosis. Moreover, chemotherapy is also an important factor playing a role in platelet activation which may affect the levels of platelet-derived microparticles in ambulatory cancer patients. Objectives: To evaluate the hypothesis that platelet-derived microparticles will increase in cancer patients compared with non-cancer patients. To extrapolate the hypothesis that levels of platelet-derived microparticles will increase after receiving chemotherapy. Methods: This was a prospective, cross-sectional study, where, we enrolled ambulatory cancer patients who were about to receive the first cycle (pre-chemotherapy) and those who already received a cycle of chemotherapy (post-chemotherapy). Controls were age- and sex- matched healthy individuals. Patients who had venous thrombosis or who were currently receiving anticoagulants were excluded. Flow cytometry was used to detect, quantify and specify platelet-derived microparticles using the co-expression of both Annexin V for phosphatidylserine and CD41a for platelet marker, and then reported as the unit of event/mL. All outcomes were compared among three groups. Results: A total of 45 patients and 15 controls were enrolled with a mean age of 58.09 years; of which, most patients were female. There was not any significant difference in age, sex, BMI, and platelet count among these groups. Gynecologic malignancy and lymphoma covered almost three-fourths of all patients, which were 51.11% and 24.44% of the total cancer patients, respectively. There was no significant difference in platelet-derived microparticles between cancer patients and controls [411,600 events/mL (331,200-722,400 events/mL) versus 563,640 events/mL (273,720-1,625,580 events/mL), p=0.3179]. Platelet-derived microparticles were significantly higher in cancer patients who had already received chemotherapy (post-chemotherapy) in comparison to cancer patients in the pre-chemotherapy group [582,000 events/mL (346,500-149,3220 events/mL) versus 402,300 events/mL (141,000 - 624,600 events/mL), p=0.0391], Figure 2. There was no significant difference in platelet-derived microparticles between hematologic and non-hematologic malignancy; however, the levels tend to be higher in the non-hematologic malignancy group. Conclusions: Platelet-derived microparticles were not significantly higher among cancer patients who have not received their first cycle of chemotherapy as compared to controls. However, after receiving chemotherapy, cancer patients exhibited significantly higher levels of platelet-derived microparticles. Chemotherapy is an important factor in platelet activation in cancer patients which may increase the levels of platelet-derived microparticles and play a role in cancer-associated thrombosis.

Presurgical circulating platelet-derived microparticles level as a risk factor of blood transfusion in patients with valve heart disease undergoing cardiac surgery

BackgroundCell-derived microparticles (MPs) are membrane vesicles that have emerged as a potential biomarker for various diseases and their clinical complications. This study investigates the role of MPs as a risk factor for blood transfusion in patients with valve heart disease undergoing cardiac surgery. MethodsForty adult patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled, and venous blood samples were collected prior to surgical incision. Plasma rich in MPs was prepared by double centrifugation, and the concentration of MPs was determined using the Bradford method. Flow cytometry analysis was performed to determine MPs count and phenotype. Patients were divided into “with transfusion” (n = 18) and “without transfusion” (n = 22) groups based on red blood cell (RBC) transfusion. ResultsThere was no significant difference in MPs concentration between the “with transfusion” and “without transfusion” groups. Although the count of preoperative platelet-derived MPs (PMPs), monocyte-derived MPs (MMPs), and red cell-derived MPs (RMPs) was higher in “without transfusion” group, these differences were not statistically significant. The preoperative PMPs count was negatively correlated with RBC transfusion (P = 0.005, r = -0.65). Multivariate logistic regression analysis revealed that the count of CD41+ PMPs, Hemoglobin (Hb), and RBC count were risk factors for RBC transfusion. ConclusionThis study suggests that the presurgical levels of PMPs, Hb, and RBC count can serve as risk factors of RBC transfusion in patients with valve heart disease undergoing cardiac surgery. The findings provide insights into the potential use of MPs as biomarkers for blood transfusion prediction in cardiac surgery.

Association of Acute Hyperglycemia and Diabetes Mellitus with Platelet-derived Microparticle (PDMP) Levels During Acute Myocardial Infarction.

This research investigates whether there is an association between acute hyperglycemia and diabetes mellitus and the level of circulating platelet-derived microparticles (PDMPs) during an initial episode of acute myocardial infarction (AMI). This was a cross-sectional study involving hospitalized AMI patients. Demographic and clinical data were obtained from hospital records. Diabetes mellitus was defined by the history of the disease, anti-diabetes medication use and/ or level of HbA1C ≥6.5%. Levels of HbA1c, admission random and fasting blood glucose levels were measured. Flow-cytometry method was used to determine the levels of PDMPs from collected venous blood through tagging with CD-41 FITC and CD-62 PE markers and a threshold size of <1 μm. The number of circulating PDMPs was compared according to glucometabolic state, namely acute hyperglycemia (admission random glucose ≥200 mg/dL and fasting glucose ≥140 mg/dL) and diabetes mellitus. The comparative analysis between groups was conducted with Student T-test or Mann-Whitney test, where applicable. A total of 108 subjects were included and their data analyzed. The level of circulating PDMPs was significantly lower in subjects with admission random glucose ≥200 mg/dL as compared to those with below level [median (interquartile range (IQR)]: 2,710.0 (718.0-8,167.0) count/mL vs. 4,452.0 (2,128.5-14,499.8) count/mL, p = 0.05) and in subjects with fasting glucose ≥140 mg/dL as compared to those with below level (median (IQR): 2,382.0 (779.0-6,619.0) count/mL vs. 5,972.0 (2,345.7-14,781.3) count/mL, p = 0.006). The level of circulating PDMPs was also significantly lower in patients with diabetes mellitus as compared to those without (median (IQR): 2,655.0 (840.0-5,821.0) count/mL vs. 4,562.0 (2,128.5-15,055.8) count/mL; p = 0.007). Acute hyperglycemia and diabetes mellitus are significantly associated with a lower circulating PDMP level during an initial AMI episode.

Differences in coagulation-relevant parameters: Comparing cryoprecipitate and a human fibrinogen concentrate.

Variable fibrinogen content within cryoprecipitate makes accurate dosing challenging in patients with coagulopathic bleeding, in addition to pathogen transmission risks associated with its administration. Purified and standardized human fibrinogen concentrates (HFCs) represent reliable alternatives. Full cryoprecipitate characterization is required to inform selection of an appropriate fibrinogen source for supplementation therapy. Extended biochemical comparison of pooled cryoprecipitate and HFC (Fibryga, Octapharma) was performed using commercially available assays to determine levels of variability in cryoprecipitate and HFC. In addition to standard procoagulant factors, measurements included activities of platelet-derived microparticles (PMPs) and plasminogen, and levels of fibrin degradation products. Cryoprecipitate contains lower fibrinogen levels than HFC (4.83 vs.19.73 g/L; p<0.001), translating to approximately half the amount of fibrinogen per standard cryoprecipitate dose (two pools, pre-pooled from five donations each) vs. HFC (2.14 vs. 3.95 g; p<0.001). Factor XIII (FXIII) levels were also lower in cryoprecipitate vs. HFC (192.17 vs. 328.33 IU/dL; p = 0.002). Levels of procoagulants in cryoprecipitate, such as von Willebrand Factor (VWF) and factor VIII (FVIII), were highly variable, as was PMP activity. A standard cryoprecipitate dose contains significantly higher levels of measured plasminogen and D-dimer fragments than a standard HFC dose. The tested HFC is a more reliable fibrinogen and FXIII source for accurate dosing compared with cryoprecipitate. Cryoprecipitate appears considerably less predictable for bleeding management due to wide variation in pro- and anticoagulation factors, the presence of PMPs, and the potential to elevate VWF and FVIII to prothrombotic levels.

Pro-Tumorigenic and Thrombotic Activities of Platelets in Lung Cancer.

Aside from their key protective roles in hemostasis and innate immunity, platelets are now recognized as having multifaceted, adverse roles in the pathogenesis, progression and outcome of many types of human malignancy. The most consistent and compelling evidence in this context has been derived from the notable association of elevated circulating platelet counts with the onset and prognosis of various human malignancies, particularly lung cancer, which represents the primary focus of the current review. Key topics include an overview of the association of lung cancer with the circulating platelet count, as well as the mechanisms of platelet-mediated, pro-tumorigenic immunosuppression, particularly the role of transforming growth factor beta 1. These issues are followed by a discussion regarding the pro-tumorigenic role of platelet-derived microparticles (PMPs), the most abundant type of microparticles (MPs) in human blood. In this context, the presence of increased levels of PMPs in the blood of lung cancer patients has been associated with tumor growth, invasion, angiogenesis and metastasis, which correlate with disease progression and decreased survival times. The final section of the review addresses, firstly, the role of cancer-related platelet activation and thrombosis in the pathogenesis of secondary cardiovascular disorders and the associated mortality, particularly in lung cancer, which is second only to disease progression; secondly, the review addresses the potential role of antiplatelet agents in the adjunctive therapy of cancer.

In-vivo functions and regulation of polyphosphate in the vascular system.

Polyphosphate, an inorganic polymer consisting of linearly linked phosphate subunits, is ubiquitously found in living organisms. Functions and regulation of the polymer have been analyzed in plants, bacteria and yeast; however, the roles of polyphosphate in mammals are still emerging. In contrast to synthetic polyphosphate that has been extensively utilized in ex-vivo studies, natural polyphosphate is complexed with bivalent cations (mostly Ca2+) and regardless of chain length, forms microparticles that are retained on the surface of procoagulant platelets, platelet-derived microparticles and cancer extracellular vesicles. On cell surfaces, these Ca2+/polyphosphate aggregates initiate the factor XII-driven contact system, triggering proinflammatory and procoagulant reactions through the kallikrein kinin system and intrinsic pathway of coagulation, respectively. Polyphosphate inhibitors interfere with thrombosis while sparing hemostasis, replicating the effect of factor XII neutralizing agents. Furthermore, polyphosphate binds to platelet factor 4, which has implications for autoimmune thrombotic diseases, such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT), potentially contributing to their pathogenesis. The metabolism and organ-specific distribution of the polymer remain incompletely defined and is the topic of ongoing research. Polyphosphate acts as a procoagulant and proinflammatory mediator. Neutralizing polyphosphate provides well tolerated thromboprotection, mimicking the effects of factor XII deficiency.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

Platelet-Derived Microparticles and Autoimmune Diseases.

Extracellular microparticles provide a means of cell-to-cell communication and can promote information exchanges between adjacent or distant cells. Platelets are cell fragments that are derived from megakaryocytes. Their main functions are to stop bleeding, regulate inflammation, and maintain the integrity of blood vessels. When platelets are activated, they can perform related tasks by secreting platelet-derived microparticles that contain lipids, proteins, nucleic acids, and even organelles. There are differences in the circulating platelet levels in many autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid antibody syndrome, and Sjogren's syndrome. In this paper, the latest findings in the research field of platelet-derived microparticles are reviewed, including the potential pathogenesis of platelet-derived microparticles in various types of immune diseases, their potential as related markers, and for monitoring the progress and prognosis of disease treatment are expounded.

CARD12: Shear Stress Renders Redistribution of P2Y12 and PAR-1 Receptors from Platelets to Microparticles: A Cue to Limited Effectiveness of Antiplatelet Therapy in MCS

Background: Platelet exposure to hypershear stress generated by mechanical circulatory support (MCS) results in platelet dysfunction, thrombosis, and bleeding coagulopathy. Antiplatelet agents, targeting ADP-evoked P2Y12 and thrombin-evoked PAR1 platelet receptors, have limited effectiveness reducing MCS thrombosis, rather favoring bleeding. Shear-mediated platelet dysfunction is associated with the downregulation of platelet adhesion receptors, decreased aggregation response, and generation of platelet-derived microparticles (PDMPs). We tested the hypothesis that MCS shear stress alters the surface expression of agonist receptors P2Y12 and PAR1 on platelets and PDMPs, thus affecting platelet aggregation response induced by ADP, collagen, and TRAP-6. Methods: Human platelets were exposed to shear stress (30-70 dynes/cm2, 10’) or sonication; PDMPs were isolated via differential centrifugation. P2Y12, PAR1, & αIIbβ3 expression on platelets and PDMPs was visualized by immunostaining and multi-colored flow cytometry. Platelets were distinguished from PDMPs by forward/side scatter vs. standard fluorescent nanobeads SPHEROTM. Platelet aggregation in plasma induced by ADP, collagen, and TRAP-6 was assessed by optical aggregometry. Results: Platelet exposure to increasing shear stress and sonication resulted in a decrease in P2Y12+ and PAR1+ platelet populations (Fig. 1A&B). The baseline surface density of P2Y12 on platelets was very low and slightly increased after 70 dyne/cm2 shear (Fig. 1C). While also low, PAR1 density on platelets tended to decrease with increasing shear stress (Fig. 1D). Shear stress induced generation of P2Y12+ and PAR1+ PDMPs, accounting for 3.5±0.6% and 4.3±0.5% after 70 dyne/cm2 shear, respectively. Remarkably, P2Y12 and PAR1 surface density on PDMPs was between 7 and 10-fold higher than on platelets (Fig. 1C&D). When added to intact platelets, PDMPs significantly decreased platelet aggregation in plasma induced by ADP, collagen, and TRAP-6. Conclusions: Elevated shear stress promotes generation of PDMPs carrying increased amounts of P2Y12 and PAR1 on their surface; the surface density of these receptors on sheared platelets was also affected. Sheared PDMPs reduced platelet aggregation likely acting as competitive inhibitors imposing steric difficulties for platelets to aggregate and thus favoring MCS bleeding phenotype and limiting the effectiveness of antiplatelet agents.

P83: Circulating Platelet-Derived Microparticles Modulate Platelet Adhesion Under Flow: Modeling Primary Thrombus Formation in a Microfluidic System

Background: Elevated levels of circulating platelet-derived microparticles (PDMPs) are associated with a range of pathologies, thought to increase thrombotic risk. We hypothesized that addition of PDMPs to whole blood would alter platelet adhesion and thrombogenicity, with its effect further modulated by shear stress magnitude. We tested the effect of addition of PDMPs to whole blood on platelet adhesion and primary thrombus formation on collagen under a range of shear flows using a microfluidic system. Methods: PDMPs were generated via sonication of gel-filtered human platelets and separated via differential centrifugation. Hirudin-anticoagulated human blood was stained with 3,3′-dihexyloxacarbocyanine iodide and circulated through a collagen-coated microchannel with and without PDMPs at defined shear stress magnitudes (13.5, 49.5, or 72 dyne/cm2) for 4 min. Platelet adhesion was visualized by fluorescence microscopy; images were analyzed using MATLAB software. Platelet adhesion and thrombogenicity was assessed via four parameters: 1. surface coverage (SC) – indicative of overall surface occupied by platelet thrombi; 2. number of thrombi (NT) – indicative of the number of sites of platelet adhesion on a collagen-covered surface; 3. mean thrombus area (MTA) and 4. mean fluorescence intensity (MFI) – both characterizing thrombus size. Results: Whole blood platelet exposure to increasing shear stress facilitated platelet adhesion to collagen, with increased SC, MTA and MFI (Fig. 1, WB group). Interestingly, the number of thrombi decreased (846±148 vs. 256±81 at 13.5 and 72 dyne/cm2, respectively) with increasing shear suggesting that increased platelet adhesion was due to increased thrombus size, rather than the number of sites of platelet adhesion. Circulating PDMPs significantly inhibited platelet adhesion to collagen, with a decrease of SC, MTI and MFI (but not NT) at all shear stress magnitudes (Fig. 1, WB+PDMPs vs. WB group, ANOVA: p<0.05). At high shear, PDMP-mediated inhibition of platelet adhesion was pronounced with a more than a 2-fold decrease of thrombus size, as indicated by MTA and MFI. Notably, adding PDMPs to recirculated whole blood increased NT adhered to collagen only at lower shear magnitudes. Conclusion: Shear stress of increasing magnitude promotes increased platelet adhesion to collagen, platelet thrombus size and surface coverage; while the number of sites of platelet adhesion decreases. Circulating PDMPs inhibit platelet adhesion under flow, resulting in a significant decrease in platelet thrombi size. Adding PDMPs to circulating blood increases the number of thrombi formed on collagen, but only at lower shear magnitudes. We speculate that increased levels of circulating PDMPs facilitate platelet adhesion and primary thrombus formation, contributing to an overall prothrombotic phenotype, yet with associated microthrombosis with embolic potential, imposing increased risk of distal ischemic and occlusive events.Figure 1. Circulating platelet-derived microparticles (PDMPs) inhibit platelet adhesion to collagen under shear stress: WB – whole blood, circulated without PDMPs; WB+PDMPs - whole blood, circulated without PDMPs (9:1). Mean ± SD, n = 7, ANOVA: * - p < 0.05, ** - p < 0.01.

Exploitation of platelets for antitumor drug delivery and modulation of the tumor immune microenvironment

Platelets are blood components traditionally believed to have fundamental roles in vascular hemostasis and thrombosis. In recent years, platelets have received new attention for their roles in tumorigenesis and progression. On the one hand, platelets are actively recruited by various tumors and comprise a crucial part of the tumor microenvironment (TME), thus inspiring the use of platelets for tumor-targeted drug delivery. To this end, various platelet-based devices have been proposed, such as natural platelets, engineered platelets, platelet membranes, and platelet-derived microparticles. On the other hand, platelets are involved in tumor immunosuppression mechanisms, by directing and/or assisting various tumor-associated immune cells. However, in the context of inflammation and autoimmune diseases, platelets can amplify immune responses by promoting immune cell mobilization and activation, thereby exacerbating tissue damage. Thus, interest is growing in the use of tumor-associated platelets as targets for therapeutic modulation of the TME and augmenting anti-tumor immune responses. In this review, we summarize current advances in exploiting platelets for both antitumor drug delivery and immune modulation of the TME.

Novel Knowledge about Molecular Mechanisms of Heparin-Induced Thrombocytopenia Type II and Treatment Targets.

Heparin-induced thrombocytopenia type II (HIT II), as stated in the literature, occurs in about 3% of all patients and in 0.1-5% of surgical patients. Thrombosis develops in 20-64% of patients with HIT. The mortality rate in HIT II has not decreased using non-heparin treatment with anticoagulants such as argatroban and lepirudin. An improved understanding of the pathophysiology of HIT may help identify targeted therapies to prevent thrombosis without subjecting patients to the risk of intense anticoagulation. The review will summarize the current knowledge about the pathogenesis of HIT II, potential new therapeutic targets related to it, and new treatments being developed. HIT II pathogenesis involves multi-step immune-mediated pathways dependent on the ratio of PF4/heparin and platelet, monocyte, neutrophil, and endothelium activation. For years, only platelets were known to take part in HIT II development. A few years ago, specific receptors and signal-induced pathways in monocytes, neutrophils and endothelium were revealed. It had been shown that the cells that had become active realised different newly formed compounds (platelet-released TF, TNFα, NAP2, CXCL-7, ENA-78, platelet-derived microparticles; monocytes-TF-MPs; neutrophils-NETs), leading to additional cell activation and consequently thrombin generation, resulting in thrombosis. Knowledge about FcγIIa receptors on platelets, monocytes, neutrophils and FcγIIIa on endothelium, chemokine (CXCR-2), and PSGL-1 receptors on neutrophils could allow for the development of a new non-anticoagulant treatment for HIT II. IgG degradation, Syk kinase and NETosis inhibition are in the field of developing new treatment possibilities too. Accordingly, IdeS and DNases-related pathways should be investigated for better understanding of HIT pathogenesis and the possibilities of being the HIT II treatment targets.