Fibrinogen Receptor Activation Research Articles

Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice

BackgroundPhosphatidylserine (PS) is a procoagulant phospholipid enriched on surfaces of activated vascular cells including platelets, endothelium, monocytes, and microvesicles. As a molecular driver of thrombosis accessible to drug blockade, PS is an attractive pharmacologic target for modulating thrombogenesis, with potentially reduced bleeding risk compared to anticoagulant and antiplatelet therapies. ObjectivesTest antithrombotic capabilities of a liposomal formulation, Zn-dipicolylamine cyanine-3[22,22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (molar ratio, 3:97), designated as DPAL, which we previously described binds selectively to PS-enriched cell surfaces, compared with effects on bleeding, in mouse models. MethodsPS-dependent DPAL binding to human and murine platelets was tested in vitro. Thrombosis and bleeding after DPAL intravenous administration were tested in C57Bl/6J mice following FeCl3 carotid arterial injury and tail tip amputation, respectively. Incorporation in hemostatic clots was investigated in the cremaster muscle laser injury model. Toxicity was tested by direct exposure to human endothelial cell cultures. ResultsDPAL bound agonist-stimulated, PS-positive human and murine platelets, blocked by Annexin V or Ano6 deletion, which ablate PS exposure. DPAL prolonged prothrombin time, but did not prevent thrombin-induced fibrinogen receptor activation or aggregation, nor alter blood cell counts including platelets. Following arteriolar laser injury, DPAL bound wound surfaces and edges without destabilizing plugs. DPAL dose-dependently blocked FeCl3-induced arterial thrombosis but did not substantially increase bleeding, or induce endothelial cell death. ConclusionDPAL reduces thrombogenesis with minimal effects on bleeding in mouse models via selective binding to PS. DPAL may support novel approaches to modulate pathogenic thrombin generation with improved safety profiles in multiple contexts.

P151 Characterization of platelet activation profile in Inflammatory Bowel Disease

Abstract Background Platelet-related markers associated with a procoagulant state have been described for Immune-mediated Inflammatory Diseases (IMIDs), but a broader profiling of these abnormalities in Inflammatory Bowel Disease (IBD) is lacking. Methods Cross-sectional study to describe and compare platelet biomarkers of IBD outpatients, both Crohn’s Disease (CD) and Ulcerative Colitis (UC), vs healthy controls (HC). Patients over 18 years old with a diagnosis of CD or UC according to the European Crohn’s and Colitis Organization (ECCO) criteria were included. Fecal calproctectin (Fcal) >=150ug/g was used as a cutoff level for active disease. Exclusion criteria for IBD patients are listed under Table 1. Fibrinogen (FBG) receptor on platelet’s surface was determined with anti-CD41 and anti-CD61 monoclonal antibodies (mAbs); and von Willebrand factor receptor with anti-CD42a and anti-CD42b mAbs. Platelet activation markers [TRAP and ADP-induced activation of fibrinogen (FBG) receptor, and P-selectin (Psel) and CD63 exposure] were determined, respectively, through binding of PAC1, anti-P-selectin and anti-CD63 (mAbs). All variables were determined by flow cytometry. Results 55 patients (27 CD, 28 UC) and 75 HC have been included. 43 IBD patients had active disease (21 CD, 22 UC). Complete baseline characteristics of the IBD population are described in Table 1. Basal Psel (% of positive cells) was increased in CD vs UC (median [IQR]: 5.64 [4,4-10,4] vs 2,9 [1,8-6,8], p=0,02) and vs HC (3,5 [1,4-6,8], p=0,008). ADP-induced Psel was increased in CD vs HC (mean, SD: 49.92, SD 12.75 vs 41.943, SD13.15, p=0,007). Basal CD63 (% of positive cells) was significantly higher in active IBD vs HC (W = 1143, p = 0.02) and inactive IBD vs HC (W = 528, p= 0.006). CD42a (% of positive cells) was decreased in IBD vs HC (W = 1128.00, p=0.007) and CD vs HC (W = 777.00, p =0.04). CD42b (% of positive cells) was also decreased in IBD vs HC (mean, SD: 106.9 SD, 17.32 vs 115.7, SD 28.42, p=0,01) and CD vs HC (104.06 SD 14.49, p=0,03). No differences in basal or TRAP/ADP induced PAC binding, and in CD41 or CD61 expression were observed among groups. Multiple linear regression showed association of Fcal with basal Psel in IBD patients (p=0,02); and Spearman’s correlation showed negative moderate correlation between Fcal and basal Psel (r=0,3; p=0,003) Conclusion Features of platelets in IBD suggest increased platelet degranulation and reduced vWF receptor with regards to healthy controls, specially in CD, but no differences in FBG-related receptors. Inverse moderate correlation between Fcal and Psel in IBD patients suggests depletion of intra-platelet granules at higher levels of gut inflammation.

Impact of Age on Hemostasis of Patients with Immune Thrombocytopenia

Introduction Incidence of primary immune thrombocytopenia (ITP) has two peaks: one in children and young adult and one in the elderly. Management of older patients represents a challenge, because older age is associated with increased frailty, comorbidities, polypharmacy, and worse outcome. Moreover, we cannot rule out the effect of old age on hemostasis of individuals without ITP. Objectives We aimed to compare characteristics of hemostasis in healthy controls and in patients with ITP stratified according to their age in ≤65 years old (yo) and >65 yo, in order to determine if old age modifies hemostasis and if there is an increased risk of bleeding in older patients with ITP. Methods This is a prospective project that was approved by the Ethics Committee from Hospital Universitario La Paz. Informed consent was signed before sampling. ITP patients (n=100≤65 yo and n=68>65 yo) and healthy controls (n=100≤65 yo and n=28>65 yo) were recruited. Patients with ITP and participants with uncontrolled hypertension, artery disease, abnormal hepatic or renal function tests, a diagnosis of a bleeding disorder or thrombopathy, treatment with drugs that could affect hemostasis or a history of thrombotic episodes were excluded. We evaluated platelet activation markers (TRAP and ADP-induced activation of fibrinogen receptor determined through PAC1-binding, and TRAP-induced P-selectin exposure with anti-P-selectin antibody); active caspase-3, -7, -8 and -9; surface loss of sialic acid determined by the binding of Ricinus Communis Agglutinin I (RCA) to galactose residues; and presence of GlcNAc residues on platelet's surface determined by the binding of Wheat Germ Agglutinin (WGA). Neuraminidase 1 (NEU1) associated to membrane of quiescent platelets were tested with anti-NEU1-Alexa 546 antibody. All samples were analyzed by flow cytometry. Thrombin generation was measured in platelet-poor plasma by Calibrated automated thrombogram (CAT) and coagulation was triggered by proper recalcification and the addition (final concentrations) of 1 pmol/l of recombinant human tissue factor and 4 µmol/l of phospholipid mixture (PPP-Reagent LOW). The following parameters were measured: Lagtime (= time when 10 nmol/l thrombin is formed); peak height (PH = maximum thrombin concentration reached); and endogenous thrombin potential (ETP = area under the thrombin-concentration-vs-time curve) were calculated with the Thrombinoscope software package (Thrombinoscope BV). Statistical analyses were performed with GraphPrism 6.0. Comparisons between controls vs ITP patients with similar range of age; controls ≤65 vs >65 yo; and ITP patients ≤65 vs >65 yo were performed with 2 way ANOVA and p<0.05 were considered as significant. Results Table 1 shows results obtained. There was no difference in age between controls and ITP groups with the same age range. Platelets from patients with ITP had a low ability to be activated when compared to their corresponding control group, and impairment in activation of fibrinogen receptor was even more evident for the older group of ITP patients.This fact was not due to a reduction in the number of fibrinogen receptors. Caspases 3, 8 and 9 were higher in ITP patients than their age-matched controls. Moreover, caspase 3 was higher in older ITP than in patients ≤65 yo. Regarding analyses of glycoside residues, platelets from patients with ITP had less sialic acid and more GlcNAc residues on their surface when compared with the corresponding controls. Surprisingly, sialic content in platelets from controls > 65yo was higher than in the controls ≤65 yo, and this fact is in accordance to the lowest content of NEU1 associated to their membrane. Plasma from ITP patients had a higher capacity to generate thrombin when compared with their age-matched control group. Nevertheless, both groups (control and ITP)> 65 yo generated less thrombin than the groups ≤65 yo. No differences were observed in other CAT parameters. Conclusion Platelets from patients with ITP are less functional than those from control groups. This impairment seemed to be more pronounced in platelets from patients >65 yo, putting forward the possibility of an increased risk of bleeding in older patients with ITP. This study also demonstrates that old age might modify hemostasis even in individuals without ITP. Work supported by Instituto de Salud Carlos III (ISCIII) (PI19/00772, PI22/01489), co-funded by the European Union.

Acetyl-CoA carboxylase 1 deletion reduces platelet phospholipid and alters thrombus formation

Acetyl-CoA carboxylase (ACC) is the key enzyme regulating de novo lipogenesis (DNL). In recent years, ACC inhibitors has been of great interest to handle a wide range of diseases, especially cancers and cardiovascular diseases. However, the impact of its inhibition on hemostasis and thrombosis remains under-investigated. We previously demonstrated that ACC1 activation promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Here, we sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions and by decreasing phospholipid content. To test this hypothesis we generated a new Cre transgenic mouse strain in which ACC1 is specifically deleted in megakaryocyte and platelet (GpIbCre±x ACC1 flx/flx mouse). Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Platelet functions were assessed in vitro by aggregometry and ex vivo by flow chamber assay. Thrombus growth was analyzed in vivo with intravital microscopy. As expected, ACC1 deletion was restricted to megakaryocytic lineage. Hematological parameters show a platelet count decrease (30%) and a platelet volume increase (31%) in ACC1 knockout mice (ACC1 pKO) compared to the control mice. In agreement with our hypothesis, lipidomics analyses highlight a significant reduction in arachidonic acid containing phosphotidylethanolamine plasmalogen in ACC1 KO platelets. Logically, thromboxane A2 generation (upon thrombin or CRP stimulation) is also altered in ACC1 pKO mice in comparison to controls. This decrease in phospholipid content is associated with a default in thrombin and CRP-induced platelet aggregation and with impaired dense granules secretion. In line with these results, platelet-specific ACC1 deletion reduces thrombus growth and fibrinogen receptor activation. Platelet-specific ACC1 deletion leads to a decrease in phospholipid content which, in turn, decreases platelet thromboxane A2 generation, dense granules secretion and platelet aggregation. Thrombus formation is also reduced in platelet-specific ACC1 knockout mice, highlighting the importance of de novo lipogenesis and ACC-produced lipids in the regulation of platelet functions.

SARS-CoV-2 Spike Protein and Neutralizing Anti-Spike Protein Antibodies Modulate Blood Platelet Function

Several studies report elevated blood platelet activation and altered platelet count in COVID-19 patients, but the role of the SARS-CoV-2 spike protein in this process remains intriguing. Additionally, there is no data that anti-SARS-CoV-2 neutralizing antibodies (nAb) may attenuate spike protein activity toward blood platelets. Our results indicate that under in vitro conditions, the spike protein increased the collagen-stimulated aggregation of isolated platelets and induced the binding of vWF to platelets in ristocetin-treated blood. The spike protein also significantly reduced collagen- or ADP-induced aggregation or decreased GPIIbIIIa (fibrinogen receptor) activation in whole blood, depending on the presence of the anti-spike protein nAb. Our findings suggest that studies on platelet activation/reactivity in COVID-19 patients or in donors vaccinated with anti-SARS-CoV-2 and/or previously-infected COVID-19 should be supported by measurements of spike protein and IgG anti-spike protein antibody concentrations in blood.

NUMBERS AND FUNCTION OF PLATELETS ARE NOT REDUCED DURING CARDIOPULMONARY BYPASS, BUT BOTH ARE REDUCED AFTER ADMINISTRATION OF PROTAMINE

<h3>Objective</h3> To assess changes in numbers and function of platelets during cardiopulmonary bypass (CPB) in CABG surgery. <h3>Methods</h3> Platelet concentration and numbers corrected for hemodilution using hemoglobin as a marker were measured before anesthesia induction (Baseline), at the end of CPB (End CPB), after administration of protamine (After protamine), at arrival at the ICU (ICU) and after 3 hours in the ICU (ICU 3h). Platelet function was assessed after activation with thrombin PAR1-receptor activating peptide (TRAP) and adenosine diphosphate (ADP), with both flow cytometry for fibrinogen receptor activation and impedance aggregometry. Flow cytometry data are presented as median fluorescence intensity (MFI), impedance aggregometry data as area under the curve in units (U). Mean ± SD and repeated measures mixed effects model together with Šídák's multiple comparisons test was used. Subsequent time points were compared to baseline. In addition, "After protamine" was compared to "End CPB". <h3>Results</h3> Thirty-nine CABG patients with mean CPB time 85.1 ± 21.3 mins were studied. During CPB platelet concentration decreased from 227 ± 48 *109/L at baseline to 179 ± 38*109/L at end of CPB (p<0.001), remained at 171 ± 41*109/L after protamine, but increased to 204 ± 48*109/L at ICU 3h (p<0.001 vs baseline). Corrected for hemodilution the number of platelets increased during CPB from 1.73 ± 0.41*109/g to 1.91 ± 0.49 *109/g (p<0.001) but decreased to 1.74 ± 0.51*109/g after protamine (p<0.01 vs End CPB), and then increased to 1.82 ± 0.48*109/g at ICU 3h (p<0.01 vs Baseline). Activation of the fibrinogen receptor, after stimulation using TRAP, did not change during CPB, but was reduced after protamine administration and lower than baseline at ICU arrival (see figure). With ADP as agonist the fibrinogen receptor activation increased from baseline 2.15 ± 1.6 to end of CPB 2.55 ± 1.6 (p<0.001) but decreased to 2.01 ± 1.3 after protamine (p<0.001 vs End CPB), returning to baseline levels at ICU 2.17 ± 1.2 and ICU 3h 2.26 ± 1.2. Platelet function measured with impedance aggregometry using TRAP as activator showed a similar pattern from baseline 114 ± 29 U, maintained levels at end of CPB 118 ± 40 U, but decreased after protamine 103 ± 34 U (p<0.05 vs End CPB), and returned to baseline levels at ICU 118 ± 32 U and finally increased at ICU 3h 126 ± 30 (p<0.05 vs Baseline). With ADP as activator the changes were similar; baseline 77.1 ± 26 U, maintained at end of CPB 68.6 ± 28 U, reduced after protamine 58.9 ± 29 U (p<0.001 vs Baseline), with return to baseline levels at ICU 66.5 ± 28 U, and ICU 3h 86.7 ± 26 U. <h3>Conclusions</h3> The number of circulating platelets increased during CPB when corrected for hemodilution. CPB did not impair platelet function. Administration of protamine reduced the function and numbers of platelets with a return to normal values at the arrival or after 3 hours in the ICU.

Predictive Value of Platelet Sequestration Studies in Splenectomy Response

Background: Splenectomy is an effective therapy for chronic immune thrombocytopenia (ITP), with high rates of long-term responses reported (about 60-80%). Because the possible related complications its indication has been limited, proving the need for further research into potential predictive factors of response. The platelet clearance determined by 111In-labelled autologous platelet scintigraphy has been proposed as an independent predictor of splenectomy outcome, with heterogenous recorded results.This study aimed to evaluate the effect of splenic platelet sequestration in predicting splenectomy response, as well as to identify other possible factors that could justify treatment failure. Methods: We retrospectively analyzed a cohort of refractory and relapsed ITP patients who underwent 111In-labelled platelet scintigraphy in one center in Madrid (La Paz University Hospital), between 2005 and 2021. The sequestration patterns were categorized based on the splenic:liver ratio (S:L), and dichotomized as splenic if purely splenic (S:L > 2) or predominantly splenic (S:L 1.4 < 2); and non-splenic if mixed (S:L 0.8 < 1.4) or hepatic (S:L < 0.8). The response to splenectomy was assessed in terms of blood platelet count at 3, 6 and 12 months post-splenectomy, according to ITP Spanish Group 2020 guidelines. Biological platelet characteristics were subsequently examined to investigate their role in splenectomy failure. Platelet activation markers (such as activation of fibrinogen receptor determined through PAC1-binding, TRAP-induced P-selectin and CD63 exposure) and active caspase-3, -7, -8 or -9 were evaluated. Platelet surface sialic acid exposure was studied by determining the binding of Ricinus Communis Agglutinin (RCA) labelled with fluorescein (FITC). Samples were analyzed by flow cytometry. Results were expressed as the ratio between each patient's values and the mean from healthy control group. Patients were separated into responders and non-responders for the analysis, applying univariate tests and multivariate regression models. Results: Platelet scintigraphy was performed in 80 patients, 51 of them underwent splenectomy. Median previous lines of treatment were 3.5 (2-6). Complications occurred in 10% of splenectomized patients, with only one non-hemorrhagic fatality event in the cohort (not related to splenectomy). Among the 51 patients with splenectomy performed, 86.2% did respond. When analyzed by sequestration pattern, complete response (CR) was observed in 75% of those with a splenic pattern. By univariate analysis, only splenic sequestration correlated with higher sustained response rate at 12 months (Table 1). The same parameter retained statistical significance through a multivariate logistic regression model (Odds ratio=37.65; p=0.005). No association was observed in terms of response with the other analyzed factors, including age, comorbidity, previous lines of treatment and previous response. Seven responders patients with splenic clearance relapsed within 3 months. Their platelet characteristics were compared to those from 16 responders controls. There were no significant differences between groups in the variables studied, except for increased binding of RCA to platelets of patients who relapsed (median, percentile 25/75= 2.05, 1.74/4.03), in contrast to those who maintained response (1.05, 0.71/1.37) (Figure 1). These findings suggest an increased desialylation pattern in platelets from the patients who relapsed. Conclusions: Our data confirm the predictive value of 111In-labelled platelet studies prior to splenectomy, with splenic sequestration emerging as an independent predictor of long-term response. Enhanced desialylation in platelets surface provides a potential explanation for splenectomy failure in those patients with a splenic clearance. The clinical relevance of these results is due to enabling a better selection of suitable candidates for splenectomy, to increase its efficacy and safety. Keywords: Chronic Immune Thrombocytopenia (ITP), splenectomy, autologous 111In-labelled platelet, splenic sequestration. Study supported by ISCIII-Fondos FEDER (PI19/00772) and PDSA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1661: THROMBOPATHY IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES AND AUTOIMMUNE DISEASE

Background: Many patients with myelodysplastic syndromes (MDS) have associated autoimmune diseases due to the presence of clonality. The presence of non specific antibodies in these patients can alter hemostasis. Aims: We aim to study if there were differences in hemostasis of patients with MDS, depending on the coexistence or not of an autoimmune disease (AD) employing global coagulation tests, and flow cytometry to evaluate platelet function. Methods: This is an observational and prospective study. We included 21 patients with MDS without any associated autoimmune disease and 15 patients with autoimmune disease (Table 1). 46 healthy controls were also recruited. The following studies were carried out: - Platelet ability to be activated was tested by flow cytometry in platelet rich plasma using TRAP and ADP to induce platelet activation, and PAC1 monoclonal antibody (mAb) to recognize activated fibrinogen receptor, and anti P- selectin and anti-CD63 mAbs to evaluate, respectively, release from alpha and dense granules. - Global assessment of clot formation by calibrated automated thrombinography (CAT) and rotational thromboelastometry (ROTEM®). Results: Platelets from MDS patients, with and without AD, showed less capacity of activation compared to the control group. Despite there were no significant differences between the groups of patients with MDS, platelets from patients without AD showed a clear decrease in their ability to be activated by TRAP and was almost absent in three of them. Kinetics of clot formation evaluated by ROTEM® showed that all MDS patients had a hypocoagulable profile. Moreover, thrombin generation was lower in patients with MDS compared to controls. We observed that in the group of patients with AD the peak of thrombin and the endogenous thrombin potential were much lower than in patients without AD (p<0.01). Accordingly, patients with AD presented a longer lag time and time to peak than patients without AD (p<0.05). Image:Summary/Conclusion: Our results suggested the presence of non-specific antibodies in plasma of patients with MDS and AD that impaired hemostasis resulting in a hypocoagulable profile.

P1656: EVALUATION OF PLATELET FUNCTION IN RARE OR UNDIAGNOSED BLEEDING DISORDERS

Background: Qualitative and/or quantitative platelet defects promote bleeding and its identification requires a careful clinical evaluation and a rational use of diagnostic laboratory assays. Aims: To study platelet function by different methods in patients with hemorrhagic symptoms who are suspected of thrombopathy to compare results obtained with each system and validate usefulness of methods employed. Methods: We included 72 patients with bleeding suspected of thrombopathy, which in some cases was confirmed by genetic study (Table 1). Patients with coagulation disorders were excluded. We used the following methods to test platelet function: Light aggregometry in platelet-rich plasma (PRP) using ADP, collagen, epinephrine and arachidonic acid as stimulating agonists. Flow cytometry in PRP using TRAP and ADP to induce platelet activation, and PAC1 monoclonal antibody (mAb) to recognize activated fibrinogen receptor, and anti P- selectin and anti-CD63 mAbs to test, respectively, release from alpha and dense granules. PFA-100 ® in whole citrated blood with collagen +ADP and collagen+Epinephrine cartridges. Total Thrombus-formation Analysis System (T-TAS ® 01, Zarcos, Japan) with T-TAS ® PL-chip (capillaries covered with collagen / under flow) in blood samples drawn in BAPA, a potent synthetic anticoagulant which inhibits Factor Xa and thrombin. Results: Some bleeding due to platelet dysfunction was diagnosed by any of the methods used, but others can only be detected with some of the techniques tested (Table 2). This difference in the sensitivity of the methods may be due to the different experimental conditions used in each of them: type of sample used, platelets’ activators, and flow vs static conditions. Forty % of undiagnosed bleedings did not seem to be related to platelet dysfunction. Image:Summary/Conclusion: All methods are complementary and their sensitivity depends on the pathway evaluated by the technique used and the mechanism involved in platelet dysfunction.

Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Influence of long-term proteasome inhibition on platelet responsiveness mediated by bortezomib

Introduction: Although platelets contain a full proteasome system, its role in platelet function is not completely understood yet. Since the proteasome system may be involved in time-delayed processes, platelet responsiveness was investigated after long-term, bortezomib-mediated proteasome inhibition.Materials and Methods: Citrate-anticoagulated whole blood was stored with 5 nM and 1 μM bortezomib for 24 h. Consecutively, aggregation was measured by light transmission in platelet-rich-plasma (PRP). Flow cytometry was performed to determine phosphorylation levels of the vasodilator-stimulated phosphoprotein (VASP), fibrinogen binding, PAC1-antibody binding and purinergic receptor expression in PRP, P2Y12 activity or glycoprotein (GP) Ib and IIb expression in whole blood. P2Y1 and P2X1 activities were assessed by calcium flux-induced fluorescence in washed platelets. Using PRP, adherent platelets on fibrinogen-, collagen- and ristocetin-coated surfaces were visualized and quantified by immunostaining.Results: Under bortezomib, VASP phosphorylation was less inducible and nitric oxide-induced inhibition of fibrinogen binding was slightly reduced. Proteasome inhibition did not tamper adenosine diphosphate-mediated aggregation or purinergic receptor expression and activity. Induced expression of activated fibrinogen receptors and fibrinogen binding were not significantly influenced by incubation with bortezomib for 24 h. Aggregation values with threshold agonist concentrations were increased under bortezomib. Despite unchanged GPIb expression, bortezomib-treated platelets showed enhanced adhesion on coated surfaces.Conclusions: In platelets incubated for 24 h, bortezomib mediates a slight attenuation of inhibitory signaling, associated with facilitated platelet aggregation using threshold agonist concentrations and enhanced adhesion on agonist-coated surfaces.

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia.

Pulmonary thrombosis is observed in severe acute respiratory syndrome coronavirus 2 pneumonia. Aim was to investigate whether subpopulations of platelets were programmed to procoagulant and inflammatory activities in coronavirus disease 2019 (COVID-19) patients with pneumonia, without comorbidities predisposing to thromboembolism. Approach and Results: Overall, 37 patients and 28 healthy subjects were studied. Platelet-leukocyte aggregates, platelet-derived microvesicles, the expression of P-selectin, and active fibrinogen receptor on platelets were quantified by flow cytometry. The profile of 45 cytokines, chemokines, and growth factors released by platelets was defined by immunoassay. The contribution of platelets to coagulation factor activity was selectively measured. Numerous platelet-monocyte (mean±SE, 67.9±4.9%, n=17 versus 19.4±3.0%, n=22; P<0.0001) and platelet-granulocyte conjugates (34.2±4.04% versus 8.6±0.7%; P<0.0001) were detected in patients. Resting patient platelets had similar levels of P-selectin (10.9±2.6%, n=12) to collagen-activated control platelets (8.7±1.5%), which was not further increased by collagen activation on patient platelets (12.4±2.5%, P=nonsignificant). The agonist-stimulated expression of the active fibrinogen receptor was reduced by 60% in patients (P<0.0001 versus controls). Cytokines (IL [interleukin]-1α, IL-1β, IL-1RA, IL-4, IL-10, IL-13, IL, 17, IL-27, IFN [interferon]-α, and IFN-γ), chemokines (MCP-1/CCL2 [monocyte chemoattractant protein 1]), and growth factors (VEGF [vascular endothelial growth factor]-A/D) were released in significantly larger amounts upon stimulation of COVID-19 platelets. Platelets contributed to increased fibrinogen, VWF (von Willebrand factor), and factor XII in COVID-19 patients. Patients (28.5±0.7 s, n=32), unlike controls (31.6±0.5 s, n=28; P<0.001), showed accelerated factor XII-dependent coagulation. Platelets in COVID-19 pneumonia are primed to spread proinflammatory and procoagulant activities in systemic circulation.

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Effects of elapsed time on downstream platelet adhesion following transient exposure to elevated upstream shear forces

Transient exposure to elevated shear forces is known to prime platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. In the present study, the platelet capture regions, prepared by immobilizing fibrinogen, collagen, or von Willebrand factor, were placed at three different distances from the upstream stenotic region to vary the elapsed time of circulating platelets downstream. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s−1 to 11,560 s−1 for all three downstream proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and the downstream capture region. In contrast, platelet adhesion to downstream collagen remained essentially independent on the distance and the adhesion to von Willebrand factor marginally increased with the distance after transient platelet exposure to upstream wall shear rates of 2145 s−1 and 11,560 s−1. The results implied that the activation of fibrinogen receptor GPIIb/IIIa by transient exposure to high upstream wall shear rates progresses in a time-dependent manner during the downstream flow of platelets. The highly elevated upstream wall shear rate of 11,560 s−1 altered the morphology of many platelets adhered to downstream fibrinogen from their native ellipsoidal to spread circular form. The platelet shape analysis showed that longer periods of post-stenotic flow increased the surface coverage fraction of ellipsoidal platelet population and decreased the surface coverage fraction of fully spread platelets on fibrinogen for both transiently elevated upstream wall shear rates.

Platelet Integrin αIIbβ3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults.

Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbβ3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbβ3 (Pearson's r= -0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= -0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50-75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbβ3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

Role of arrestin1 in regulation of GPCR signaling in platelet activation

The arrestins are a class of soluble proteins that function in concert with the GPCR kinases to stop or arrest GPCR‐mediated intracellular signaling. Although β‐arrestins function in the desensitization of most GPCRs, the functional differences of β‐arrestin1 (arrestin2, BARR1) versus β‐arrestin2 (arrestin1, BARR2) in the regulation of GPCR signaling in platelets and the mechanisms of desensitization of platelets to GPCR agonists by β‐arrestins have not been understood. In this study, we identify the role of BARR2 in the regulation of platelet function and GPCR signaling in platelets. We used mice lacking BARR1 and BARR2 to evaluate the functional role of BARR2 in platelet activation. Platelet aggregation, dense‐granule secretion, and fibrinogen receptor activation induced by 2‐MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in BARR2 −/− platelets compared to WT platelets. In contrast to BARR2 −/− platelets, AYPGKF‐, thrombin‐, and U46619‐induced platelet aggregation and secretion were significantly inhibited in BARR1 −/− platelets compared to WT platelets, while 2‐MeSADP‐induced platelet aggregation and secretion were minimally affected in BARR1 −/−platelets. However, CRP‐induced platelet aggregation and secretion were not affected in both BARR1 −/− and BARR2 −/− platelets. These results suggest that BARR2 functions as the predominant regulator in the termination of GPCR signaling in platelets. In addition, platelet aggregation was restored in response to second challenge of 2‐MeSADP and AYPGKF in BARR2 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT and b‐arrestin1 −/− platelets, indicating BARR2 contributes to P2Y1, P2Y12, and PAR receptor desensitization. Furthermore, 2MeSADP‐ and AYPGKF‐induced Akt and ERK phosphorylations were significantly potentiated in BARR2 −/− platelets. Finally, BARR2 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating BARR2 is critical for thrombus formation in vivo. We conclude that BARR2 plays an important role in regulation of platelet function through selective GPCR desensitization.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).

Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors

Heparin and protamine are fundamental in the management of anticoagulation during cardiac surgery. Excess protamine has been associated with increased bleeding. Interaction between protamine and platelet function has been demonstrated but the mechanism remains unclear. We examined the effect of protamine on platelet function in vitro using impedance aggregometry, flow cytometry, and thrombin generation. Platelets were exposed to protamine at final concentrations of 0, 20, 40, and 80 µg/mL, alone or together with adenosine diphosphate (ADP) or thrombin PAR1 receptor-activating peptide (TRAP). We found that in the absence of other activators, protamine (80 µg/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% (p < .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 µg/mL reduced aggregation, from 73.8 ± 29.4 U to 46.9 ± 21.1 U (p < .001) with ADP and from 126.4 ± 16.1 U to 94.9 ± 23.7 U (p < .01) with TRAP. P-selectin exposure (a marker of alpha-granule release) measured by median fluorescence intensity (MFI) increased dose dependently with protamine alone, from 0.76 ± 0.20 (0 µg/mL) to 10.2 ± 3.1 (80 µg/mL), p < .001. Protamine 80 µg/mL by itself resulted in higher MFI (10.16 ± 3.09) than activation with ADP (2.2 ± 0.7, p < .001) or TRAP (5.7 ± 2.6, p < .01) without protamine. When protamine was combined with ADP or TRAP, there was a concentration-dependent increase in the alpha-granule release. In conclusion, protamine interacts with platelets in vitro having both a direct activating effect and impairment of secondary activation of aggregation by other agonists.

Effects of Elapsed Time on Downstream Platelet Adhesion Following Exposure to Transiently Elevated Upstream Shear Forces

Transient exposure to elevated shear forces pre-activates platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. We found that platelet adhesion to downstream capture region containing one of the three immobilized platelet binding proteins: fibrinogen, collagen, or von Willebrand factor, changed after platelets flowed through an upstream stenotic region. These platelet capture regions were placed at three different distances from the upstream stenotic region. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s-1 to 11560 s-1 for all three proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and downstream capture region. Platelet adhesion to collagen remained essentially independent on the distance and adhesion to von Willebrand factor marginally increased with the distance after transient platelet exposure to higher upstream shear rates. The results implied that the activation of fibrinogen receptor GPIIb/IIIa by transient exposure to high upstream wall shear rates progresses in a time-dependent manner during the flow of platelets to the downstream capture regions. The highly elevated upstream wall shear rate of 11560 s-1 altered the morphology of many platelets adhered to downstream fibrinogen from their native ellipsoidal to spread circular form. The platelet shape analysis showed that longest periods of post-stenotic flow increased the surface coverage fraction of ellipsoidal platelet population and decreased the surface coverage fraction of fully spread platelets on fibrinogen after both elevated upstream wall shear rates.

Prothrombotic State, Platelet Activation and Netosis in Systemic Lupus Erythematosus

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown origin characterized by a hypercoagulable state and a high mortality rate. Mechanisms that cause the accelerated deterioration of cardiovascular health in SLE are unknown. Objectives: to characterize the prothrombotic state in SLE patients by global coagulation assays and the contribution of platelets, endothelial damage, microparticles and neutrophil extracellular traps (NETs) in their prothombotic profile. Material and methods: 72 patients and 90 healthy controls were recruited. Patients were classified according to clinical characteristics in: 32 with lupus (SLE group), 29 with SLE and antiphospholipid antibodies (aFL, SLE+aFL group) and 12 who met the criteria for SLE and antiphospholipid syndrome (APS, SLE+APS group). Experimental protocol was approved by La Paz University Hospital Ethics Committee. Venous blood collected in BD sodium citrate tubes (3.2%) was centrifuged at 150 g for 20 min at 23ºC to obtain platelet-rich plasma (PRP). PPP was obtained by centrifugation at 1500 g for 15 min at 23ºC. To obtain neutrophils, whole blood was centrifuged to 1600 rpm 25 min using a Ficoll gradient and red cells were lysed. Rotational thromboelastometry (ROTEM®) was performed in naTEM condition. Clotting time (CT, time from start of measurement until initiation of clotting [in seconds]); alpha angle (tangent to the curve at 2-mm amplitude [in degrees]), Ax (clot firmness at time x, [in mm]) and maximum clot firmness (MCF, [in mm]) were recorded. Procoagulant activity associated to microparticle's content of tissue factor was determined in PPP by Calibrated Automated Thrombogram (CAT) using MP-reagent (4 mM phospholipids, Diagnostica Stago, Spain). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced) and the time to the peak. Thrombin generation associated to NETs was also measured by CAT. Neutrophils from healthy controls or from LES patients were stimulated with 100 nM PMA in RPMI medium during 45 min at 37º and then cocultivated with PRP adjusted to 105 platelets/µL. NETs formation was verified by fluorescent microscopy performed with DAPI and an anti-myeloperoxidase antibody. Plasma levels of LDL-ox, E-Selectin and PAI-1 were determined by Elisa (R&amp;D Systems, MN, USA and Affymetrix eBioscience, Vienna, Austria, respectively). Platelet activation was analysed by flow cytometry (FCM, FACScan, BD Biosciences). Fibrinogen receptor activation was evaluated through PAC1-FITC binding and release of granule's content was assessed with monoclonal antibodies (mAbs) anti-CD63 and anti P-selectin in quiescent and 100 µM TRAP and 10 µM ADP stimulated platelets. Data were analysed with Graphpad prism and p ≤0.05 was stablished as statistical significance. Results: PAI-1 plasma level was increased in all patient's groups, whereas LDL-ox and E-selectin showed no differences with control cohort (Fig.1). ROTEM demonstrated a procoagulant profile in SLE and SLE+aPL but not in SLE+APS group (Fig. 2). PAI-1 levels correlated with several ROTEM parameters (Table 1). SLE patients and SLE+aFL showed a basal platelet activation. Moreover, SLE group exposed more P-selectin and CD63 than controls (Fig.3). Regarding thrombin generation associated to tissue-factor content of microparticles, no differences were observed between SLE patients and healthy controls. On the other hand, SLE patients had an increased peak of thrombin generation related to NETs formation (control group: 170.3± 58.0, SLE patients: 230.6±39.3, p=0.019). Conclusions: ROTEM® detected a hypercoagulable state in SLE and SLE+aPL patients. The hypercoagulable state might be linked to increased PAI-1 plasma levels and basal platelet activation in SLE and SLE+aPL groups. Moreover, neutrophils from SLE patients seemed to present a basal activation that induced a NETs-related procoagulant state in these patients. SLE+APS patients did not show a hypercoagulable state perhaps because of the presence of lupus anticoagulant and/or to therapeutic treatment of these patients. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Fernandez-Bello: Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Robles:ABBVIE, SANDOZ FARMACEUTICA: Speakers Bureau. Álvarez Roman:Sobi: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau. Canales:Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Speakers Bureau; Sandoz: Honoraria; iQone: Honoraria; Takeda: Speakers Bureau; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Varying effects of tyrosine kinase inhibitors on platelet function-A need for individualized CML treatment to minimize the risk for hemostatic and thrombotic complications?

Since their introduction, tyrosine kinase inhibitors (TKIs, eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib) have revolutionized the treatment of chronic myeloid leukemia (CML). However, long‐term treatment with TKIs is associated with serious adverse events including both bleeding and thromboembolism. Experimental studies have shown that TKIs can cause platelet dysfunction. Herein, we present the first side‐by‐side investigation comparing the effects of currently used TKIs on platelet function and thrombin generation when used in clinically relevant concentrations. A flow cytometry multiparameter protocol was used to study a range of significant platelet activation events (fibrinogen receptor activation, alpha granule, and lysosomal exocytosis, procoagulant membrane exposure, and mitochondrial permeability changes). In addition, thrombin generation was measured in the presence of TKIs to assess the effects on global hemostasis. Results show that dasatinib generally inhibited platelet function, while bosutinib, nilotinib, and ponatinib showed less consistent effects. In addition to these general trends for each TKI, we observed a large degree of interindividual variability in the effects of the different TKIs. Interindividual variation was also observed when blood from CML patients was studied ex vivo with whole blood platelet aggregometry, free oscillation rheometry (FOR), and flow cytometry. Based on the donor responses in the side‐by‐side TKI study, a TKI sensitivity map was developed. We propose that such a sensitivity map could potentially become a valuable tool to help in decision‐making regarding the choice of suitable TKIs for a CML patient with a history of bleeding or atherothrombotic disease.