Differences In Platelet Activation Research Articles

Effect of Freeze-Thawing Treatment on Platelet-Rich Plasma Purified with Different Kits.

Osteoarthritis of the knee (OAK), a progressive degenerative disease affecting quality of life, is characterized by cartilage degeneration, synovial inflammation, and osteophyte formation causing pain and disability. Platelet-rich plasma (PRP) is an autologous blood product effective in reducing OAK-associated pain. PRP compositions depend on their purification. In clinical practice, PRP is typically administered immediately after purification, while cryopreserved PRP is used in research. Platelets are activated by freezing followed by release of their humoral factors. Therefore, PRP without any manipulation after purification (utPRP) and freeze-thawed PRP (fPRP) may differ in their properties. We purified leukocyte-poor PRP (LPPRP) and autologous protein solution (APS) to compare the properties of utPRPs and fPRPs and their effects on OAK target cells. We found significant differences in platelet activation and humoral factor content between utPRPs and fPRPs in both LPPRP and APS. Freeze-thawing affected the anti-inflammatory properties of LPPRP and APS in chondrocytes and synovial cells differed. Both utPRPs and fPRPs inhibited polarization toward M1 macrophages while promoting polarization toward M2 macrophages. Freeze-thawing specifically affected humoral factor production in macrophages, suggesting that evaluating the efficacy of PRPs requires considering PRP purification methods, properties, and conditions. Understanding these variations may enhance therapeutic application of PRPs in OAK.

High-Dimensional Single-Cell Mass Cytometry Demonstrates Differential Platelet Functional Phenotypes in Infants With Congenital Heart Disease.

Congenital heart disease (CHD) is a group of complex heart defects associated with hematologic abnormalities, including increased risk of thrombotic and bleeding events. Past studies have observed evidence of platelet hyperreactivity, while other studies showed decreased platelet activation in patients with CHD. The goal of this study was to develop a mass spectrometry approach to characterize single platelets in infants with CHD and identify potential etiology for such discrepant results. We enrolled 19 infants with CHD along with 21 non-CHD controls at Yale New Haven Children's Heart Center. A single-cell high-dimensional mass cytometry method was developed to quantitatively interrogate platelet surface markers in whole blood. Additionally, plasma cytokine analysis was performed through a multiplexed panel of 52 vascular and inflammatory markers to assess for platelet releasates. We found that infants with CHD had significant differences in platelet activation and functional markers by mass cytometry compared with non-CHD controls. Based on cell surface markers, we classified the platelets into 8 subpopulations (P0 to P7). Distinct subpopulations of platelets (P1, P4, and P5) exhibiting decreased aggregatory phenotype but altered secretory phenotypes were also identified and found to be more abundant in the blood of infants with CHD. Electron microscopy identified increased proportion of hypogranular platelets in CHD. Moreover, cytokine analysis demonstrated an overall increase in plasma cytokines and biomarkers in CHD, including IL (interleukin)-6, IL-8, IL-27, RANTES, and VWF (von Willebrand factor), which are expressed in platelet granules and can be released upon activation. We developed a robust mass cytometry approach to identify platelet phenotypic heterogeneity. Infants with CHD had alterations in distinct subpopulations of platelets with overall reduced aggregatory phenotype and secretory dysfunction. These findings suggest that platelets in infants with CHD may be exhausted due to persistent stimulation and may explain both bleeding and thrombotic vascular complications associated with CHD.

Cutibacterium acnes contamination does not enhance the proinflammatory profile of platelet concentrates.

Cutibacterium acnes, a common anaerobic platelet concentrate (PC) contaminant, has been associated with rare mild adverse transfusion reactions and is often considered a harmless commensal. Notably, C. acnes can cause chronic infections and has been shown to induce the release of proinflammatory cytokines by immune cells. Since elevated concentrations of proinflammatory factors in PCs have been linked to noninfectious adverse reactions, this study aimed to assess whether C. acnes could elicit the release and accumulation of proinflammatory factors during PC storage, thereby enhancing the risk of such reactions. Four ABO-matched buffy coat PCs were pooled and split into six units, each were inoculated with either saline (negative control), a Staphylococcus aureus isolate (positive control, 30 colony forming units [CFU]/unit), or four C. acnes PC isolates (10 CFU/mL) and stored at 20-24°C with agitation. Bacterial counts, platelet activation, and concentration of proinflammatory factors were assessed on days 0, 3, and 5. N = 3. C. acnes counts remained stable, while S. aureus proliferated reaching 108CFU/mL by the end of PC storage. By day 5, no significant differences in platelet activation or proinflammatory cytokine profiles were observed in C. acnes-contaminated PCs compared to the negative control (p > .05), while there was a significant increase (p ≤ .05) in sCD40L concentration (day 3), and platelet activation and IL-8 concentration (day 5) in S. aureus-contaminated units. C. acnes contamination does not promote the accumulation of proinflammatory factors in the absence of proliferation during storage and may not enhance the risk of inflammatory reactions when transfused to patients.

Separation of platelets by size in a microfluidic device based on controlled incremental filtration.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.

The bleeding diathesis in patients with hereditary haemorrhagic telangiectasia is not due to impaired platelet function

BackgroundPatients with the rare disease; Hereditary haemorrhagic telangiectasia (HHT) often bleed from telangiectatic lesions in mucosal surfaces. Studies suggest that impaired platelet function may also play a role in their bleeding tendency. The aim of the present study was to investigate whether HHT-patients with epistaxis have impaired platelet function.MethodWe conducted a case–control study based on a sample size calculation and included 22 HHT-patients (inclusion criteria: epistaxis severity score ≥ 4, no intake of medicine affecting platelet function the last 5 days, HHT-type 1 or 2, age ≥ 18 years) and 20 controls. We assessed the platelet function with standard haemostasis parameters, flow cytometry (platelet function and micro aggregation), rotational thromboelastometry and Platelet Function Analyzer 200.ResultsWe found no significant difference in mean platelet volume and immature platelet fraction and no difference in platelet activation as measured by exposure of CD62P, CD63P and PAC1 binding. Nor did we find a significant difference in platelet aggregation response in HHT-patients compared with the control group for all agonists (thrombin receptor activating peptide, adenosine diphosphate and collagen-related peptide). The PFA-200 analysis was without difference between the two groups and thromboelastometry showed no impairment of global haemostasis.ConclusionReduced platelet function is unlikely to contribute to the frequent and long bleeding episodes that HHT-patients suffer from. We propose that further studies should focus on whether patients with HHT have hypercoagulability.

FRI114 PUFAs Implicated In Platelet Activation Differ In Receptor Mediated Vs Post-receptor Insulin Resistance

Abstract Disclosure: R. Klein: None. S.T. Chung: None. R.J. Brown: None. Background: Severe insulin resistance (IR) can be classified as receptor mediated, in which all downstream signaling is blocked, or post receptor IR where some downstream signaling pathways are decreased and others are increased. Receptor-mediated IR occurs in rare mutations of the insulin receptor (INSR), whereas in most forms of IR, including type 2 diabetes (T2DM) and lipodystrophy (LD), IR is post-receptor. LD is a group of syndromes characterized by deficiency of fat depots leading to metabolic dysfunction similar to obesity. Patients with post-receptor IR have increased atherosclerotic cardiovascular disease (ASCVD) risk. Thrombosis underlies pathology of ASCVD and is a result of platelet (PLT) activation. Polyunsaturated fatty acids (PUFAs) are embedded in the PLT membrane and play a key role in thrombosis. During PLT activation, cytosolic phospholipase A2 (cPLA2) hydrolyzes membrane lipids, generating free PUFAs, leading to synthesis of mediators of blood vessel constriction, inflammation, and thrombosis. Methods: This was a cross-sectional study of subjects with INSR (N=30) and LD (N=30) matched for age, sex, HbA1c and pubertal stage. Untargeted metabolomic (848 compounds) and lipidomic (984 compounds) analysis was completed on serum after an 8-12 hour fast. Analyses were conducted by t-test and Mann-Whitney with adjustment for multiplicity. Bonferroni corrected P<0.05 was considered significant. Results: Of 19 long chain PUFAs (ω=3, ω=6) measured, 14 were significantly elevated in LD vs INSR, including arachidonate, docosahexaenoate, docosapentaenoate, stearidonate, and adrenate. There was no difference in linoleate, dihomo-linoleate, tetradecadienoate, linolenate, or docosadienoate levels between LD vs INSR. Among 8 long chain saturated FAs, only 3 were significantly higher in LD, 4 were comparable, and 1 was higher in INSR. All long and medium chain monounsaturated FAs were similar in LD vs INSR. cPLA2 was not different between LD and INSR. Conclusions: Our study shows that liberated PUFAs were higher in the patients with LD compared to INSR. A similar trend was not seen for other FFA such as saturated and monounsaturated FFA, suggesting that higher PUFAs in LD are unlikely to be caused by global differences in FFA metabolism, such as different peripheral uptake of FFA via lipoprotein lipase activity or diet. Instead, our data suggest an increase in PLT membrane ability to store and release PUFAs. These data suggest increased ASCVD risk in post-receptor insulin resistance may be partially mediated through differences in platelet activation. Presentation: Friday, June 16, 2023

IRGM/Irgm1 deficiency inhibits neutrophil-platelet interactions and thrombosis in experimental atherosclerosis and arterial injury

BackgroundNeutrophil extracellular traps (NETs) closely link inflammation and thrombosis. The immune-related GTPase family M protein (IRGM) and its ortholog of mouse IRGM1 are positively correlated with plaque rupture during atherosclerosis process. However, whether and how IRGM/IRGM1 affects NETs formation and atherosclerotic thrombosis remains unknown, which will further promote the development of antithrombotic treatment tools. MethodsThe thrombi images, platelet activation makers and NETs makers were detected in the serum of STEMI patients and controls. To futher investigate IRGM/IRGM1 affects NETs formation and atherothrombosis in vivo, ApoE-/-Irgm1+/- and ApoE-/- mice received diets rich in fat and 2.5% FeCl3 was then used to induce experimental arterial thrombosis in an atherosclerosis background. In vitro, PMA and thrombin were used to stimulate neutrophils and platelets, respectively, and the expression of IRGM/IRGM1 were modified. To reveal the molecular mechanisms, MAPK-cPLA2 signals inhibitors were used. ResultsSerum IRGM was positively correlated with PF4 and neutrophil elastase. Subsequently, Irgm1 deficient mice have a longer occlusion time and lower growth rate. In vitro, as expected, IRGM/Irgm1 deficiency inhibits platelet activation and platelet-neutrophil interaction. More importantly, IRGM promoted NETs production through activating MAPK-cPLA2 signals in PMA stimulated neuropils, whereas inhibiting the production of NETs eliminated the difference in platelet activation and thrombosis caused by IRGM/Irgm1 modification in vivo and vitro. Similarly, inhibition of platelet activation also eliminated the influence of IRGM/Irgm1 modification on NETs production. ConclusionsOverall, our data indicate that IRGM/Irgm1 deficiency in neuropils inhibits the intense interaction between neutrophils and platelets, and ultimately inhibits thrombosis.

Sex-related differences in the response of anti-platelet drug therapies targeting purinergic signaling pathways in sepsis.

Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high mortality. Sex-related differences in the immune response to sepsis have been proposed but the mechanism is still unknown. Purinergic signaling is a sex-specific regulatory mechanism in immune cell physiology. Our studies have shown that blocking the ADP-receptor P2Y12 but not P2Y1 receptor was protective in male mice during sepsis, but not female. We now hypothesize that there are sex-related differences in modulating P2Y12 or P2Y1 signaling pathways during sepsis. Male and female wild-type (WT), P2Y12 knock-out (KO), and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. The P2Y12 antagonist ticagrelor or the P2Y1 antagonist MRS2279 were administered intra-peritoneally after surgery to septic male and female mice. Blood, lungs and kidneys were collected 24 hours post-surgery. Sepsis-induced changes in platelet activation, secretion and platelet interaction with immune cells were measured by flow cytometry. Neutrophil infiltration in the lung and kidney was determined by a myeloperoxidase (MPO) colorimetric assay kit. Sepsis-induced platelet activation, secretion and aggregate formation were reduced in male CLP P2Y12 KO and in female CLP P2Y1 KO mice compared with their CLP WT counterpart. Sepsis-induced MPO activity was reduced in male CLP P2Y12 KO and CLP P2Y1 KO female mice. CLP males treated with ticagrelor or MRS2279 showed a decrease in sepsis-induced MPO levels in lung and kidneys, aggregate formation, and platelet activation as compared to untreated male CLP mice. There were no differences in platelet activation, aggregate formation, and neutrophil infiltration in lung and kidney between female CLP mice and female CLP mice treated with ticagrelor or MRS2279. In human T lymphocytes, blocking P2Y1 or P2Y12 alters cell growth and secretion in vitro in a sex-dependent manner, supporting the data obtained in mice. In conclusion, targeting purinergic signaling represents a promising therapy for sepsis but drug targeting purinergic signaling is sex-specific and needs to be investigated to determine sex-related targeted therapies in sepsis.

O079 Predictive value for elevated platelet-derived SGPVI in venous in-stent stenosis in patients with post-thrombotic syndrome

Abstract Introduction In-stent stenosis following intervention for post-thrombotic syndrome (PTS) occurs in ∼30% of cases, despite therapeutic anticoagulation. Our aim was to investigate whether platelets are involved. Methods Blood was taken from case-matched patients before and after venous stenting. Flow-cytometry was used to assess platelet activation (P-selectin and/or phosphatidylserine exposure) while= functional activity was measured by plate-based aggregation and through a flow-cell model of thrombus formation. Soluble glycoprotein VI (sGPVI) was measured in plasma. Patients with in-stent stenosis requiring reintervention (>50% diameter reduction) were compared with those who did not. Results Forty-five patients were recruited (median age: 43yrs (33–55yrs); 65% female), and 19/45 (42%) required reintervention (median time: 3wks (1day-3mths)). There was no difference in platelet activation or reactivity after stenting, but P-selectin exposure pre-stent was significantly higher in patients who developed in-stent stenosis (2.7%±0.4 vs 1.7%±0.2; P<0.05). Pre-stent sGPVI levels were increased in patients who developed in-stent stenosis (18.9±3.6ng/mL vs. 7.4±0.9ng/mL; P<0.01). Platelet reactivity to collagen-related peptide, a GPVI-specific platelet agonist, was reduced in patients who developed in-stent stenosis (logEC50 = -6.5M±0.3 vs -7.2M±0.2; P<0.05; n=33). Pre-stent platelet accumulation (26.6AU±0.5 vs 19.0AU±8.1; P=0.04) was significantly increased in PTS patients compared with healthy controls. Conclusion Venous stenting does not activate platelets, but patients who developed in-stent stenosis exhibited greater levels of pre-stent platelet activation, greater loss of platelet surface GPVI in the form of sGPVI and reduction in reactivity to GPVI activation. sGPVI may have potential to risk stratify patients undergoing venous stenting and predict who requires closer surveillance. Take-home message Soluble GPVI may have potential to risk-stratify patients undergoing deep venous reconstruction and predict who requires closer surveillance.

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study.

Background and AimsPlatelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function.MethodsFourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high‐frequency acceleration data logger. Shock index scores were calculated as shock intensity (g‐force) times frequency.ResultsThe shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor‐activating peptide‐6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes.ConclusionThough the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport.

Platelet Function in Thrombocytopenic Patients with Chronic Liver Disease

Background: Thrombocytopenia, the most common hematologic abnormality encountered in patients with chronic liver disease (CLD), results from multiple factors including splenic sequestration, relatively reduced production of thrombopoietin for the degree of thrombocytopenia, consumption of platelets secondary to autoantibodies directed against platelets, and bone marrow suppression. Elevated plasma levels of von Willebrand factor (VWF) in CLD patients may partly compensate for the reduced platelet count via interaction of VWF with the platelet receptors glycoprotein (GP) Ib-IX-V and GPIIb-IIIa (integrin αIIβ3), to promote platelet adhesion and aggregation following vessel injury. Platelets in patients with CLD are reported to have reduced function as measured by light transmission platelet aggregation (LTA) and other assays. However, LTA and most other platelet function tests are unreliable in thrombocytopenic samples. In contrast, platelet function evaluated by whole blood flow cytometry is accurate in samples with low platelet counts. Therefore, in the present study we used whole blood flow cytometry to determine whether platelet function as measured by platelet surface P-selectin, activated GPIIb-IIIa and GPIb on in vivo -circulating and in vitro -stimulated platelets is altered in thrombocytopenic CLD patients compared to healthy controls.Methods: Platelet function end points were evaluated in untreated CLD patients with thrombocytopenia (n = 21) participating at U.S. sites in the Phase 3 E5501-G000-310 or E5501-G000-311 studies of a novel thrombopoietin receptor agonist, avatrombopag, compared to healthy controls (n=10). IRB approval was obtained at each study site and written informed consent was obtained from each subject. CLD patient samples analyzed in the present study were collected prior to the administration of any study drug. No patient received a platelet transfusion within 10 days of the blood draw for this study. Platelet surface P-selectin, activated GPIIb-IIIa and GPIb, without or with in vitro stimulation (0.5 μM adenosine diphosphate [ADP], 20 μM ADP, 1.5 μM thrombin receptor activating peptide [TRAP] or 20 μM TRAP) were measured by whole blood flow cytometry. To explore whether the differences in these markers between CLD patients and healthy subjects were related to the difference in platelet count rather than to differences in platelet activation or reactivity, whole blood from healthy controls was diluted with platelet-poor plasma to adjust the platelet counts to those of the thrombocytopenic CLD patients.Results: Circulating platelets (i.e., no in vitro stimulation) from thrombocytopenic CLD patients showed low levels of platelet surface P-selectin and activated GPIIb-IIIa, similar to the healthy subject controls, demonstrating minimal in vivo platelet activation in thrombocytopenic CLD patients. However, the platelets of thrombocytopenic CLD patients had higher platelet surface GPIb mean fluorescence intensity (MFI) than either diluted or undiluted circulating platelets from healthy controls (Figure). In vitro stimulation of CLD patient platelets by ADP or TRAP resulted in lower levels of platelet surface P-selectin and activated GPIIb-IIIa compared to healthy controls, except for the levels of surface P-selectin after stimulation with 20 μM TRAP. The results for the diluted healthy subject controls were unaffected by the platelet count, except for the samples activated with 1.5 μM TRAP in which dilution resulted in reduced platelet surface P-selectin and activated GPIIb-IIIa.Conclusions: 1) Circulating platelets in thrombocytopenic CLD patients are not activated. 2) Agonist-stimulated platelet surface expression of P-selectin and activated GPIIb-IIIa is reduced in thrombocytopenic CLD patients compared to healthy controls with the same degree of thrombocytopenia, which likely contributes to the excessive bleeding in thrombocytopenic CLD patients. 3) Thrombocytopenic CLD patients have a higher expression of GPIb on circulating platelets compared with healthy controls with the same degree of thrombocytopenia. This finding is consistent with the presence of an increased proportion of large immature platelets. The high levels of platelet surface GPIb, together with the previously reported high levels of circulating VWF in these patients, may contribute to vascular integrity at lower platelet counts. [Display omitted] DisclosuresFrelinger:Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; Baxalta: Research Funding; GLSynthesis: Research Funding; Ionis: Research Funding. Michelson:GLSynthesis: Research Funding; Eisai: Research Funding; Ironwood: Research Funding; Sysmex: Research Funding; Ionis: Research Funding; Pfizer: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Elsevier: Patents & Royalties; Baxalta: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Instrumentation Laboratory: Consultancy.

The effects of additive solutions on the development of storage lesions in stored canine platelet concentrates.

To determine if platelet additive solutions (PAS) decrease the occurrence and degree of platelet storage lesions, maintain platelet function, and extend storage time in vitro beyond 5 days at 22°C when compared to platelets stored in plasma only. Prospective, ex vivo experimental controlled study. Research laboratory in a school of veterinary medicine. Twelve units of canine platelet concentrate prepared from fresh whole blood donations. Platelet concentrates were aliquoted into 4 units and stored at room temperature (22°C) under constant agitation in either 100% plasma (control) or 35% plasma and 65% of 1 of 3 different PAS (Plasma-Lyte A, Isoplate, and InterSol) for 7 days. At days 0, 3, 5, and 7, samples were analyzed for presence of swirling, degree of aggregate formation, platelet count, platelet indices, glucose, lactate, lactate dehydrogenase, Pvo2 , and Pvco2 concentrations, aggregation via light aggregometry, and activation percentage based on flow cytometric measurement of surface P-selectin. Bacterial cultures were performed on days 0, 5, and 7. Isoplate had a higher incidence of aggregate formation on day 0 (n=2), and Plasma-Lyte A had a higher incidence of loss of swirl on day 7 (n=5). Plasma-stored samples had significantly higher platelet counts (P< 0.001), pH (P< 0.05), Pvco2 (P< 0.001), and lactate (P< 0.001), and significantly lower lactate dehydrogenase (P< 0.05) as compared to all PAS. The mean pH remained above 7.2 in PAS and plasma. There was no difference in platelet activation between plasma and PAS. Changes in platelet indices, glucose consumption, and maximum aggregation varied by storage solution. There was no bacterial growth seen in any samples. The 3 PAS performed similarly and could all be considered as potential replacements for plasma during the room temperature storage of canine platelet concentrate for up to 7 days.

Feasibility study of use of rabbit blood to evaluate platelet activation by medical devices

It is important to ascertain platelet responses to blood-contacting medical devices as part of a complete hemocompatibility evaluation. Nevertheless, researchers often face the problem of insufficient quantities of human blood for evaluation of platelet activation by actual medical devices. If animal blood can replace human blood to evaluate platelet activation by medical devices, testing will be smoother and will aid for quality control of related products. Therefore, in this study, we exposed representative biomaterials to human blood, rabbit blood and mouse blood, and evaluated similarities and differences in platelet activation among the three types of blood by measuring various molecular markers. We found that rabbit blood and human blood had considerable similarity in terms of platelet activation, while mouse blood and human blood showed considerable differences. Therefore, rabbit blood may replace human blood for platelet function testing.

Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

Evaluating platelet activation related to the degradation of biomaterials using molecular markers

The effective assessment of platelet activation is an important component of the evaluation of cardiovascular implants. Currently, most evaluation is performed based on the ISO 10993-4 international standard. However, the methods specified in this standard were originally designed for non-degradable materials, and the applicability of these methods to evaluate degradable materials has not been carefully assessed. Here, the platelet activation response was evaluated (using blood from health rabbits) for three typical degradable materials (collagen, polylactic acid, and hydroxyapatite) by measuring the widely used molecular markers CD62 P, CD63, and CD40 L and the three molecular markers PF4, β-TG, and TXB2 that are referenced in the ISO 10993-4 standard. The variations of these six markers were compared in the simulated degradation of the three test materials. The results showed differences in platelet activation with degradation that were strongly related to the surface physicochemical properties. Changes in the surface roughness and contact angle of the materials correlated with changes in the degree of platelet activation. The six tested platelet activation molecular markers show promise for assessment of platelet function in degradable medical devices, providing guidance for quality control strategies and the design and improvement of safe medical devices.

Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice

AbstractDeficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Use of a Cyclooxygenase-2 Inhibitor Does Not Inhibit Platelet Activation or Growth Factor Release From Platelet-Rich Plasma

Background: It remains unestablished whether use of cyclooxygenase (COX)–2 inhibitors impairs platelet activation and anabolic growth factor release from platelets in platelet-rich plasma (PRP). Purpose: The purpose of this study was to assess the effects of a COX-2 inhibitor on platelet activation and anabolic growth factor release from canine PRP when using a clinically applicable PRP activator and to determine whether a 3-day washout would be sufficient to abrogate any COX-2 inhibitor-related impairment on platelet function. Study Design: Controlled laboratory study. Methods: Ten healthy dogs underwent blood collection and PRP preparation. Dogs were then administered a COX-2 inhibitor for 7 days, after which PRP preparation was repeated. The COX-2 inhibitor was continued for 4 more days and PRP preparation performed a third time, 3 days after discontinuation of the COX-2 inhibitor. Immediately after PRP preparation, the PRP was divided into 4 aliquots: 2 unactivated and 2 activated using human γ-thrombin (HGT). One activated and 1 unactivated sample were assessed using flow cytometry for platelet expression of CD62P and platelet-bound fibrinogen using the canine activated platelet-1 (CAP1) antibody. The 2 remaining samples were centrifuged and the supernatant assayed for transforming growth factor–β1 (TGF-β1), platelet-derived growth factor–BB (PDGF-BB), and thromboxane B2 (TXB2) concentrations. Differences in platelet activation and TGF-β1, PDGF-BB, and TXB2 concentrations over the 3 study weeks were evaluated using a 1-way repeated-measures ANOVA, and comparisons between activated and unactivated samples within a study week were assessed with paired t tests. Results: There were no statistically significant (P > .05) effects of the COX-2 inhibitor on percentage of platelets positive for CD62P or CAP1 or on concentrations of TGF-β1, PDGF-BB, or TXB2. All unactivated samples had low levels of activation or growth factor concentrations and significantly (P < .05) greater activation and growth factor concentrations in HGT-activated samples. Conclusion: This COX-2 inhibitor did not impair platelet activation, growth factor release, or TXB2 production in this canine PRP when using HGT as an activator. Studies are warranted to determine whether COX-2 inhibitors affect platelet activation and growth factor release from human PRPs. Clinical Relevance: These results suggest that there is no need to withhold a COX-2 inhibitor before PRP preparation, particularly if thrombin is going to be used to activate the PRP. This is clinically relevant information because many patients who are candidates for PRP therapy for treatment of musculoskeletal injury are also using COX-2 inhibitors.

Glutamate Receptor Interacting Protein 1 Mediates Platelet Adhesion and Thrombus Formation.

Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium.

Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbβ3 expression and clot retraction in platelets

Integrin αIIbβ3 is critical for platelet-mediated blood clotting. Tetraspanins are well-established regulators of integrins and genetic loss of tetraspanin CD151 or TSSC6 in mice leads to increased bleeding due to inadequate integrin αIIbβ3 outside-in signaling. Conversely, mild but enhanced integrin αIIbβ3 activation and hyperaggregation is observed in CD9 and CD63 null mice respectively. CD82 is reportedly expressed in platelets; however its function is unknown. Using genetically engineered CD82 null mice, we investigated the role of the tetraspanin CD82 in platelet activation. Loss of CD82 resulted in reduced bleed times in vivo. CD82 was present on the surface of both human and mouse platelets, and its levels did not change upon platelet activation or degranulation. No differences in platelet activation, degranulation, or aggregation in response to ADP or collagen were detected in CD82 null mice. However, the kinetics of clot retraction was enhanced, which was intrinsic to the CD82-null platelets. Integrin αIIbβ3 surface expression was elevated on the platelets from CD82 null mice and they displayed enhanced adhesion and tyrosine kinase signaling on fibrinogen. This is the first report on CD82 function in platelets; which we found intrinsically modulates clot retraction, integrin αIIbβ3 expression, cell adhesion, and tyrosine signaling.