Alpha-granule Release Research Articles

Abstract 154: Hierarchical Redundancy And Contextual Roles Of Vesicle-associated Membrane Proteins (VAMPs) In Platelet Function

Background: Platelet granule secretion, driven by the SNARE complex involving Vesicle-Associated Membrane Proteins (VAMPs) and t-SNAREs, is essential for vascular integrity. We dissected individual and combinatorial contributions of all four platelet VAMPs to granule secretion, hemostasis, and intracellular cargo trafficking. Research Questions: 1. Are specific VAMPs critical for granule fusion, or does total VAMP quantity (mass action) dictate secretion? 2. How do differential VAMP deficiencies impact hemostasis in arterial and venous microenvironments? 3. Do VAMPs influence platelet cargo loading and gene expression? Goals: Investigate individual and combinatorial roles of VAMPs in platelet biology and their context dependent contributions to hemostasis. Approach: We generated V7 -/- 8 -/- and V2 Δ 3 Δ 7 -/- 8 -/- mice, assessing granule secretion (dense, alpha, lysosomal) via ATP, PF4/P-selectin, and β-hexosaminidase/LAMP1 release assays, respectively. Hemostasis in arterial and venous models was evaluated with tail bleeding, FeCl3, and venous puncture injury assays. Cargo levels were analyzed using proteomic arrays, and platelet RNA sequencing identified transcriptional changes. Results: Global V7/V8 deletion modestly reduced secretion compared to isolated V8 deficiency. Surprisingly, while platelet-specific V2 Δ 3 Δ 7 -/- 8 -/- exhibited the most dramatic decrease, complete abolition was absent (p<0.01). Alpha and lysosomal secretion were more affected than densegranule release. Both knockout models bled profusely and failed to form stable arterial thrombi (p<0.01). Notably, only V2 Δ 3 Δ 7 -/- 8 -/- mice displayed significantly prolonged venous bleeding (p<0.01). RNA sequencing revealed >800 upregulated and >500 downregulated transcripts in V7 - /- 8 -/- and V2 Δ 3 Δ 8 -/- platelets, suggesting shared transcriptional control by V7 and secondary VAMPs (V2 and V3). Conclusions: VAMP functions exhibit hierarchical redundancy, with V8 presence minimally impacting dense granule secretion and arterial hemostasis. Alpha granule release is more sensitive to VAMP loss. The requirements for platelet granule secretion for hemostasis are context dependent. The deletion of VAMPs disrupts platelet cargo trafficking and transcription.

Mitochondrial Fission Protein Drp1 Regulates Platelet Function

Background: Mitochondria in platelets contribute to platelet function and are altered by diseases that involve dysregulated hemostasis or thrombosis. Mitochondrial integrity, governed by its size, number and distribution is modulated by fusion and fission proteins. Pharmacological inhibition of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) has been shown to inhibit granule release in human platelets and impair thrombus formation in a murine model of thrombosis. However, the mitochondrial roles for Drp1 in platelet function are still largely unknown. Objective: To determine the mitochondrial roles of Drp1 in regulating platelet function. Methods: PF4-Cre mediated MK/platelet specific Drp1 null mice (Drp1 -/-) were used. Platelet morphology in Drp1 -/- and wild type (WT) platelets was analyzed by confocal microscopy. Procoagulant platelet formation (PS +ve platelets) after thrombin and the GPVI agonist convulxin (THR+CVX) stimulation was measured using Annexin V staining. Mitochondrial potential (ΔΨm) and mass at baseline and after stimulation was measured using TMRM and mitotracker green. Washed platelet activation in WT and Drp1 -/- platelets in response to convulxin was measured by flow cytometry. Platelet cytoplasmic calcium transients were measured using FURA-2AM after stimulation with THR+CVX. Phosphorylation of Drp1 (pDrp1 S616) in washed WT platelets in response to dual agonist THR+CVX was measured by western blotting. Results: Drp1 -/- platelets were bigger in size than WT platelets as measured by microscopy and had increased mitochondrial mass as determined by flow cytometry. Absence of Drp1 reduced activation of the αIIb/β3 fibrinogen binding receptor (P=0.035; n=5-6 per group) and reduced alpha granule release (P=0.025; n=5-6 per group) in response to CVX.Dual agonist stimulation resulted in reduced PS +ve platelets in Drp1 -/- compared to WT platelets (P=0.0035; n=7 per group). Since depolarization precedes PS exposure, we measured ΔΨm using TMRM in resting and dual agonist stimulated WT and Drp1 -/- platelets. No difference in ΔΨm was observed in resting Drp1 -/- vs WT platelets whereas, stimulated Drp1 -/- platelets exhibited significantly higher ΔΨm (P=0.038; n=4 per group) than stimulated WT platelets. Interestingly, in response to dual agonists, Drp1 -/- platelets also showed a significant increase (P<0.001) in cytoplasmic calcium transients than WT platelets. Western blot analyses of washed WT platelets stimulated with dual agonist showed phosphorylation of Drp1 at S616 (S616), indicating Drp1 activation. Conclusion: These findings highlight a role for Drp1 in murine platelet mitochondrial function, GPVI activation, and procoagulant platelet formation.

Vagus nerve stimulation primes platelets and reduces bleeding in hemophilia A male mice

Deficiency of coagulation factor VIII in hemophilia A disrupts clotting and prolongs bleeding. While the current mainstay of therapy is infusion of factor VIII concentrates, inhibitor antibodies often render these ineffective. Because preclinical evidence shows electrical vagus nerve stimulation accelerates clotting to reduce hemorrhage without precipitating systemic thrombosis, we reasoned it might reduce bleeding in hemophilia A. Using two different male murine hemorrhage and thrombosis models, we show vagus nerve stimulation bypasses the factor VIII deficiency of hemophilia A to decrease bleeding and accelerate clotting. Vagus nerve stimulation targets acetylcholine-producing T lymphocytes in spleen and α7 nicotinic acetylcholine receptors (α7nAChR) on platelets to increase calcium uptake and enhance alpha granule release. Splenectomy or genetic deletion of T cells or α7nAChR abolishes vagal control of platelet activation, thrombus formation, and bleeding in male mice. Vagus nerve stimulation warrants clinical study as a therapy for coagulation disorders and surgical or traumatic bleeding.

Uncoupling of platelet granule release and integrin activation suggests GPIIb/IIIa as a therapeutic target in COVID-19.

Thromboembolic events are frequent and life-threating complications of COVID-19, but are also observed in patients with sepsis. Disseminated thrombosis can occur despite anticoagulation, suggesting that platelets play a direct, but yet incompletely understood role. Several studies demonstrated altered platelet function in COVID-19 with in part controversial findings, while underlying disease-specific mechanisms remain ill-defined. We performed a comprehensive cohort study with 111 patients, comprising 37 with COVID-19, 46 with sepsis, and 28 with infection, compared to controls. Platelet phenotype and function were assessed under static and flow conditions, revealing unexpected disease-specific differences. From hospital admission on, platelets in COVID-19 failed to activate integrin GPIIb/IIIa in response to multiple agonists. Dense granule release was markedly impaired due to virtually missing granules, also demonstrated by whole mount electron microscopy. In contrast, alpha-granule marker CD62P exposure was only mildly affected, revealing a subpopulation of PAC-1-/CD62P+ platelets, independently confirmed by automated clustering. This uncoupling of alpha-granule release was not observed in sepsis patients, despite a similar disease severity. We found overall unaltered thrombus formation in COVID-19 and sepsis samples under venous shear rates, which was dependent on the presence of tissue factor. Unexpectedly, under arterial shear rates thrombus formation was virtually abrogated in sepsis, while we detected overall normal-sized and stable thrombi in blood from COVID-19 patients. These thrombi were susceptible to subthreshold levels of GPIIb/IIIa blockers eptifibatide or tirofiban that had only a minor effect in control blood. We provide evidence that low dose GPIIb/IIIa blockade could be a therapeutic approach in COVID-19.

Loss of P2Y1 receptor desensitization does not impact hemostasis or thrombosis despite increased platelet reactivity in vitro

BackgroundThe hemostatic plug formation at sites of vascular injury is strongly dependent on rapid platelet activation and integrin-mediated adhesion and aggregation. However, to prevent thrombotic complications, platelet aggregate formation must be a self-limiting process. The second-wave mediator adenosine diphosphate (ADP) activates platelets via Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors. After ADP exposure, the P2Y1 receptor undergoes rapid phosphorylation-induced desensitization, a negative feedback mechanism believed to be critical for limiting thrombus growth. ObjectiveThe objective of this study was to examine the role of rapid P2Y1 receptor desensitization on platelet function and thrombus formation in vivo. MethodsWe analyzed a novel knock-in mouse strain expressing a P2Y1 receptor variant that cannot be phosphorylated beyond residue 340 (P2Y1340-0P), thereby preventing the desensitization of the receptor. ResultsP2Y1340-0P mice followed a Mendelian inheritance pattern, and peripheral platelet counts were comparable between P2Y1340-0P/340-0P and control mice. In vitro, P2Y1340-0P/340-0P platelets were hyperreactive to ADP, showed a robust activation response to the P2Y1 receptor-selective agonist, MRS2365, and did not desensitize in response to repeated ADP challenge. We observed increased calcium mobilization, protein kinase C substrate phosphorylation, alpha granule release, activation of the small GTPase Rap1, and integrin inside-out activation/aggregation. This hyperreactivity, however, did not lead to increased platelet adhesion or excessive plug formation under physiological shear conditions. ConclusionOur studies demonstrate that receptor phosphorylation at the C-terminus is critical for P2Y1 receptor desensitization in platelets and that impaired desensitization leads to increased P2Y1 receptor signaling in vitro. Surprisingly, desensitization of the P2Y1 receptor is not required for limiting platelet adhesion/aggregation at sites of vascular injury, likely because ADP is degraded quickly or washed away in the bloodstream.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1β/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Investigation of the Relevance of Platelet Alpha Granules for Postoperative Liver Regeneration in a Mouse Model of Partial Hepatectomy

Introduction: Platelet alpha granule release was previously associated to development of liver dysfunction after liver resection. However, the relevance of platelet degranulation in liver regeneration has not been elucidated thus far. Here we aimed to investigate differences in liver regeneration between mice lacking alpha granules and wild type individuals after partial hepatectomy (pHx).

The Alpha-Granule Release Reaction Is Mediated By SNARE-Dependent Solubilization of the Granule Matrix

Within their electron dense granule matrix, alpha-granules package a wide range of proteins important to hemostasis, thrombosis, inflammation, and angiogenesis. During the platelet release reaction, these proteins are secreted in a kinetically diverse manner that is proportional to agonist stimulation. The release reaction is accompanied by a large expansion in granule volume, matrix decondensation, and the solubilization of the included proteins. We have previously posited that some combination of differential protein proximity to granule exit sites and protein solubilization could contribute to the diversity of protein release.Here we have used thrombin stimulation and SNARE knockout mice to test two possible mechanisms for granule decondensation/matrix solubilization. In the first, granule swelling and decondensation precedes fusion with the plasma membrane and is regulated by aquaporin-mediated water influx presumably driving the volume change. In the second, matrix solubilization is plasma membrane fusion dependent with the extracellular influx of water through the fusion pore driving volume change and granule decondensation.In wild-type mouse platelets, we found a precursor-product relationship between the incidence of condensed and decondensed granules with the incidence of decondensed granules peaking at 90 sec post-stimulus (0.1 U/mL thrombin). The subsequent decrease in decondensed granules was due to an increasing incidence of compound granule fusion. By electron tomography, granules were connected to the plasma membrane by “necks” or short “pipes”. The granule membrane remained physically distinct during the entire granule decondensation process. Granule decondensation was marked by the conversion of the granule matrix into a diffuse fibrous structure that decreased progressively in electron density over the 5 min stimulation period. A residual electron-dense core was most obvious at 90 sec post stimulation. At the plasma membrane fused granule neck/pipe, solubilized granule matrix fibers were observed to be extruded extracellularly.Overall, the data suggest a progressive solubilization of granule matrix contents and their accompanying extrusion through the granule neck/pipe. In SNARE knockout mouse platelets, SNARE-selective inhibition of matrix solubilization/granule decondensation was observed. Both qualitatively and quantitatively, knockout of VAMP2/3 had little-to-no effect on 1) alpha-granule fusion with the plasma membrane, 2) granule decondensation, 3) matrix solubilization, or 4) compound granule fusion. In striking contrast, VAMP8 knockout initially produced a substantial inhibition of the incidence of all four steps in the granule releasate reaction while a VAMP2/3/8 knockout almost totally inhibited all four steps. In sum, our data strongly indicate that alpha-granule decondensation and matrix protein solubilization/extrusion are highly dependent on SNARE-mediated granule fusion with the plasma membrane. These data provide no support for the alternative mechanism that regulated aquaporin-dependent water influx mediates granule decondensation prior to granule fusion with the plasma membrane or canalicular system.This work was supported in part by NIH grant R01 HL 119393 to BS, NIH grant R01 HL56652 to SWW, and AHA grant 15PRE2555020 to SJ. DisclosuresNo relevant conflicts of interest to declare.

Bruton’s Tyrosine Kinase Inhibitors Impair Fcγriia-Mediated Responses of Healthy Donor Platelets and Chronic Lymphocytic Leukaemia Derived Platelets to Bacteria

Platelets play a key role in innate immunity and can interact with bacteria through multiple molecular mechanisms. One key receptor for platelet-bacteria interactions is FcγRIIa, a low affinity IgG immune receptor found on the surface of platelets, which is associated with platelet aggregation, phagocytosis and the release of bactericidal substances and cytokines from alpha and dense granules. The signalling pathways that regulate FcγRIIa mediated platelet responses to bacteria are not fully understood. Downstream of the FcγRIIa receptor is the protein Bruton's tyrosine kinase (Btk) and potentially other Tec family kinases, yet the role of these kinases in platelet-bacteria interactions is unknown. Btk is a therapeutic target in chronic lymphocytic leukaemia (CLL) with inhibitors of Btk (iBtks) including ibrutinib and acalabrutinib. However, iBtks have off-target effects on platelets, inhibiting aggregation with associated haemorrhagic side effects. iBtk treatment is also associated with a higher incidence of infection in CLL patients. The effect of iBtks on platelet immune function has not been evaluated. We hypothesise that Btk has a role in platelet FcγRIIa signalling in response to bacterial agonists, and that iBtks inhibit such responses, contributing towards the increased risk of infection seen with such therapies in CLL. We show that ibrutinib and acalabrutinib inhibit healthy donor FcγRIIa-mediated platelet aggregation, alpha and dense granule release in response to incubation with Staphylococcus aureus and Escherichia coli, and also in response to FcγRIIa crosslinking with the monoclonal antibody IV.3 (anti-FcγRIIa). The observed lack of granule secretion will reduce the bactericidal substances released by the platelet, as well as the amount of cytokine and chemokine secretion limiting cross-talk with other immune cells. Phosphorylation of Btk at tyrosine 223 (a marker of Btk activation) was detected in response to FcγRIIa agonists, and was inhibited by both ibrutinib and acalabrutinib. Little is known about the effect of iBtk treatment on CLL-platelet responses to bacteria. CLL patients tend to be on concurrent medications for comorbid conditions that could alter platelet responses. We show that treatment-naïve and ibrutinib-treated CLL platelets have aggregation and alpha granule release to thrombin receptor activator peptide 6 and ADP comparable to healthy controls, indicating that CLL platelet responses to these FcγRIIa-independent agonists are normal. Moreover, platelets derived from iBtk naïve CLL patients aggregate normally to bacteria in the presence of autologous plasma. However, platelets from ibrutinib-treated CLL patients have significantly inhibited aggregation and alpha granule release in response to S.aureus, E.coli, and IV.3 crosslinking. Moreover, Btk is phosphorylated at Y223 in response to bacterial agonists in treatment-naïve, but not in ibrutinib-treated CLL platelets, highlighting the lack of Btk activation in iBtk-treated patients. An X-linked agammaglobulinaemia (XLA) patient with a known Btk loss of function mutation was examined to investigate if Btk is vital for the FcγRIIa pathway. XLA-derived platelets aggregated normally in response to multiple bacterial species, suggesting Btk is redundant in mediating platelet aggregatory responses to bacteria. These results suggest that other Tec family kinases might have a role in platelet FcγRIIa activation to bacteria, which could be affected by iBtk treatment too. In conclusion, we propose that iBtks impair the FcγRIIa pathway in platelets, reducing platelet-bacteria responses, possibly contributing to the increased risk of infections in observed in CLL. Disclosures No relevant conflicts of interest to declare.

Blood group-related antigen Ley on human platelets and its involvement in platelet aggregation via a possible interaction with CD61.

The Ley antigen is a carbohydrate chain belonging to the ABH-Lewis blood group family. Ley has been reported to be present on red blood cells (RBCs) and granulocytes, but its distribution and function in platelets remain unknown. There are a variety of glycoproteins on platelets, which may carry the Ley antigen. This study aims to investigate the expression pattern and the function of Ley in human platelets. Flow cytometry, Western blot, and immunofluorescence assays were performed to determine Ley expression on human platelets. ADP (1.25-10 μM) and thrombin (0.05-1 IU/mL) were used to activate platelets in the presence or absence of prostaglandin E1 (PGE1) and the Ley expression was evaluated again by flow cytometry. Blockade was performed with an anti-Ley monoclonal antibody to verify the role of this epitope in platelet function. Finally, coimmunoprecipitation was performed to identify glycoproteins associated with Ley . Ley was expressed on human platelets independent of ABO blood type. Ley expression was decreased in a dose-dependent manner after activation with either ADP or thrombin, and this effect could be partially reversed by PGE1. Anti- Ley mAb treatment increased alpha-granule release and neutralized the inhibitory effect of the anti-CD61 antibody on platelet aggregation. In addition, Ley was proven to interact and colocalize with CD61. These results demonstrate nondifferential expression of Ley in platelets of different ABO blood types and suggest the involvement of Ley in platelet function, possibly via interaction with CD61.

Pharmacologic dissection of the overlapping impact of heat shock protein family members on platelet function

BackgroundPlatelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis. ObjectivesThe role of heat shock proteins (Hsps) in modulating platelet function has been studied for a number of years, but comparative roles of individual Hsps have not been thoroughly examined. MethodsWe utilized a panel of specific inhibitors of Hsp40, Hsp70, Hsp90, and Grp94 (the endoplasmic reticulum homolog of Hsp90) to assess their impact on several aspects of platelet function. ResultsInhibition of each of the aforementioned Hsps reduced alpha granule release. In contrast, there was some selectivity in impacts on dense granule release. Thromboxane synthesis was impaired after exposure to inhibitors of Hsp40, Hsp90, and Grp94, but not after inhibition of Hsp70. Both expression of active glycoprotein IIb/IIIa (GPIIb/IIIa) and fibrinogen‐induced platelet shape change were diminished by our inhibitors. In contrast, aggregation was selectively abrogated after inhibition of Hsp40 or Hsp90. Lastly, activated platelet–cancer cell interactions were reduced by inhibition of both Hsp70 and Grp94. ConclusionsThese data suggest the importance of Hsp networks in regulating platelet activity.

Stepping Up to the Plate(let) against Candida albicans.

Candida albicans is a pervasive commensal fungus that is the most common pathogen responsible for invasive fungal infection (IFI). With incidence of IFI on the rise due to increasing susceptible populations, it is imperative that we investigate how Candida albicans interacts with blood components. When stimulating either human or mouse whole blood with thrombin, we saw a significant decrease in C. albicans survival. We then repeated Candida killing assays with thrombin-stimulated or unstimulated washed platelets and saw a similar decrease in CFU. To investigate whether killing was mediated through surface components or releasable products, platelets were pretreated with an inhibitor of actin polymerization (cytochalasin D [CytoD]). CytoD was able to abrogate C. albicans killing. Moreover, dilution of releasates from thrombin-stimulated platelets showed that the toxicity of the releasates on C. albicans is concentration dependent. We then investigated C. albicans actions on platelet activation, granule release, and aggregation. While C. albicans does not appear to affect alpha or dense granule release, C. albicans exerts a significant attenuation of platelet aggregation to multiple agonists. These results illustrate for the first time that platelets can directly kill C. albicans through release of their granular contents. Additionally, C. albicans can also exert inhibitory effects on platelet aggregation.

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.

Analysis of Platelet Alpha and Dense Granule Release Using Small Volumes of Blood

A significant limitation of the gold-standard platelet function test, light transmission aggregometry (LTA), is the relatively large volume of blood required, as well as need for a platelet count in the resulting platelet-rich plasma of not much less than 100,000/μL, thus potentially precluding this test in pediatric and thrombocytopenic patients. Flow cytometric assessment of platelet activation in diluted whole blood avoids these limitations, typically employing anti-CD62P (P-Selectin) binding reflecting alpha granule release and PAC-1 binding reflecting conversion of GPIIb/IIIa to its active conformation. We have recently reported the use of tandem mass spectrometry to assess platelet dense granule release of deuterated serotonin in comparably diluted whole blood samples (https://doi.org/10.1093/ajcp/aqz094). In the present study we examine the possibility of adapting and optimizing the luciferin-luciferase (L-L) approach often used together with LTA for the assay of platelet dense granule ATP release from 10-fold diluted whole blood in parallel with flow cytometric analysis. We optimized the L-L reaction with commercially available reagents and used a conventional lumi-aggregometer to measure real-time secretion of platelet dense granules following agonist stimulation in 10-fold diluted whole blood obtained from healthy adult volunteers, with or without specimen stirring at 1000 RPM. Direct comparison of optimized reagents to a commercial L-L reagent kit showed an improved lower limit of detection for exogenously added ATP by at least one order of magnitude. Initial studies were performed under non-stirring conditions. Dose-dependency of agonist was observed, with representative results for released ATP of 243.1 ± 29.7 pmol/107 platelets in response to 40 μM TRAP (thrombin receptor-activating peptide) and 18.9 ± 2.4 pmol/107 platelets in response to 1 nM convulxin (GPVI receptor activator) (mean ± SEM, N = 15). In contrast, virtually no ATP secretion was observed in response to 10 μM ADP or to 10 μg/mL collagen. Despite the quite low platelet count (as low as 11,000/μL to date) following the 10-fold dilution of whole blood, introducing 1000 RPM stirring to these samples now permitted robust ATP release in response to collagen as low as 2 μg/mL. There continued to be little or no ATP release, however, in response to 10 μM ADP even with the addition of stirring. Flow cytometry was used to interrogate agonist-stimulated alpha granule release and GPIIb/IIIa conformation change. Following incubations with agonist and fluorescently-labeled antibodies, the 10-fold diluted whole blood was further diluted an additional 10-fold, and gating based on light scatter and binding of anti-CD61 antibody used to identify individual platelets. Under non-stirring conditions, TRAP, ADP, and convulxin all produced strong P-selectin exposure, as well as conversion of GPIIb/IIIa to its active conformation. Analysis of samples that had undergone 1000 RPM stirring during incubation with agonist showed quite similar results in response to TRAP. While still strongly positive, the responses to ADP in the stirred specimens showed decreases typically of at least 50%. Without stirring, platelet responses to 2-10 μg/mL collagen were virtually absent, whereas in the stirred specimens significantly positive anti-CD62P and PAC-1 binding were now observed. The combined flow cytometric and L-L studies required &lt;1 mL blood. The results demonstrate the dramatic differences observed in the release of platelet alpha and dense granules, depending upon conditions and assay systems employed. We have now shown that with agonist addition to the same 10-fold diluted whole blood samples employed in flow cytometric platelet analysis, dense granule release may also be assessed by modifications of the L-L methodology already employed for lumi-aggregation studies in many laboratories. We have additionally shown that by employing stirring of the specimen during incubation with agonist, collagen itself may be employed as platelet stimulus, rather than having to rely upon convulxin. The difference in requirements for alpha versus dense granule release, however, remains exemplified by ADP: While a potent stimulus of alpha granule release, ADP employed in the 10-fold diluted whole blood setting appeared incapable of producing significant dense granule release either in the absence or presence of sample stirring. Disclosures Wool: Diagnostica Stago: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

Platelet Isolation and Activation Assays.

Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, including secretion of soluble bioactive molecules via the release of alpha and dense granules, activation of membrane adhesion receptors, release of microparticles, and externalization of phosphatidylserine. These processes facilitate firm adhesion to sites of injury and the recruitment and activation of other platelets and leukocytes, resulting in aggregation and thrombus formation. Platelet activation drives the hemostatic response, and also contributes to pathogenic thrombus formation. Thus, quantification of platelet-associated responses is key to many pathophysiologically relevant processes. Here we describe protocols for isolating, counting, and activating platelets, and for the rapid quantification of cell surface proteins using flow cytometry.

Abstract 52: IkB Kinase beta is a Key Modulator of Platelet Function, and the Remodeling of CARMA1-Bcl10-MALT1 Complex Formation

Secretion plays an important role in platelet function, and hence the secretory machinery offers a unique target to modulate thrombogenesis. We previously established that IκB kinase (IKK)-β is phosphorylated upon platelet activation, regulates their SNARE machinery, and involves CBM (CARMA1, Bcl10, and MALT1) complex formation. However, the detailed role of IKKβ in platelet function, the mechanism by which it regulates CBM complex formation and downstream effector activation remain elusive. Using a knockout mouse model system ( IKK β flox/flox -PF4Cre), we first showed that IKKβ plays a vital role in thrombus formation, and that it does so, in part, by regulating platelet functional responses, including dense and alpha granule release, αIIbβ3 activation, and PS exposure. Furthermore, we observed defects in compound fusion, platelet spreading, actin remodeling, and clot retraction. To this end, under clot retraction conditions in the knockout platelets, 7S complex formation was found to be inhibited. In terms of its signaling, using knockout platelets, we observed that IKKβ is required for the recruitment of Bcl10/MALT1 to CARMA1 upon platelet activation, i.e., CBM complex formation, and that this was due to the defective phosphorylation of Bcl10 and the IKKγ polyubiquitination. In conclusion, our data shows that IKKβ is a key regulator of platelet activation, in vitro and in vivo , and the remodeling of the CBM complex. Furthermore, our findings indicate that inducible clustering of signaling mediators and the formation of higher-order multi-protein complexes (i.e., the CBM complex) is a dynamic process, that supports nonlinear signaling networks and is important for platelet activation.

Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice

Background/Aims: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. Methods: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. Results: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin α_IIbβ₃ on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. Conclusions: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.

Flow Cytometry Protocols for Assessment of Platelet Function in Whole Blood.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.

The Common PAR4 Ala120Thr Variant Has a Major Effect on Platelet Reactivity to Thrombin and These Effects Are Enhanced with PAR1 and P2Y12 Inhibition

▪Introduction: Human platelets express two thrombin receptors, protease activated receptor (PAR) 1 and PAR4 with different affinities for thrombin and different signaling properties. PAR1 and PAR4 have non-identical tissue expression patterns and repertoire of protease activators. We have shown a significant difference in activation kinetics and calciummobilization between platelets from healthy white and black subjects stimulated with PAR4-AP (Nat Med 2013). Part of these racial differences appears to be mediated by PAR4 signaling through Gq to Rap1 and PKC (ATVB 2014). Given the importance of platelet thrombus formation in ischemic arterial disease, these findings may contribute to the known worse outcomes in blacks compared to whites after acute coronary syndromes. Genomic approaches demonstrated that ~50% of the racial variance in PAR4-AP-induced platelet aggregation and calcium flux is caused by an Ala120Thr substitution in PAR4 (encoded by rs773902) and that the Thr120 variant is the "hyperreactive" compared to Ala120 (Blood 2014). Importantly, the Thr120 allele frequency is 63% in blacks and 19% in whites. However, the effect of the physiologic activator, thrombin, on this platelet PAR4 variant (independent of race) has not been characterized. The goals of this work were to perform a detailed characterization of the PAR4 Ala120Thr effect on thrombin-induced platelet function, and to assess pharmacogenetic effects of the PAR4 Ala120Thr variant on standard anti-platelet agents and novel PAR4 inhibitors.Results:We recruited 130 healthy, multi-racial donors and genotyped for rs773902. Subjects were excluded who had reduced arachidonic acid aggregation or who had the rare PAR4 Val296 variant. Preliminary studies showed heterozygotes had intermediate phenotypes to all assays, so most comparisons were between washed platelets homozygous for Thr120 or Ala120. The thrombin dose response curve for Thr120 homozygotes was left-shifted relative to Ala120 homozygotes (p=0.022 for genotype effect; n=10 per genotype). The concentration of thrombin required to produce 50% maximal aggregation (ED50) was 17% lower for Thr120 homozygotes than for Ala120 homozygotes (0.041 ± 0.002 U/mL vs. 0.049 ± 0.002 U/mL, respectively; p<0.001). The PAR4-AP dose response for Thr120 homozygotes was also left-shifted compared to Ala120 homozygotes (p<0.0001 for genotype effect; n=11 per genotype), as would be expected for functional variants in PAR4. The PAR4-AP ED50 was 2.5-fold lower for Thr120 homozygotes than for Ala120 homozygotes (41.0 uM ± 5.0 uM vs. 101.0 ± 4.5 uM respectively; p<0.001) Compared to Ala120 homozygotes, Thr120 homozygote platelets showed higher thrombin-induced ATP release and P-selectin exposure, indicating higher levels of dense and alpha granule release, and higher calcium mobilization, indicating Gq coupling (n=4-7 per genotype).Next, washed platelets were incubated with varying concentrations of YD-3 (PAR4 blocker), ASA (COX inhibitor), 2-MeSAMP (P2Y12 antagonist) or the PAR1 antagonist, vorapaxar, and assessed for aggregation after stimulation with 0.1 U/mL thrombin. The concentration of vorapaxar required to inhibit 50% of maximal platelet aggregation (IC50) was 2.6 fold lower for Ala120 homozygotes relative to Thr120 homozygotes (45.5 ± 7.6 nM vs. 118.6 ± 40.9 nM, p<0.0001; n=3 per genotype). The IC50 of 2-MeSAMP was substantially lower for Ala120 homozygotes relative to Thr120 homozygotes (4.0 ± 5.0 uM vs. 10.9 ± 4.8 uM, p=0.056; n=4 per genotype). YD-3 and ASA were unable to inhibit thrombin-induced aggregation regardless of the PAR4 variant.Conclusions: These data indicate that the common Ala120Thr variant alters platelet reactivity to physiologic agonists, that the Thr120 variant has decreased sensitivity to PAR1 and P2Y12 inhibition, and predict the PAR4 Thr120 variant would alter the risk-benefit of PAR1 inhibition. Since current antiplatelet therapy is based largely on platelet studies done with white subjects, novel PAR4 inhibitors may provide improved anti-thrombotic therapy for patients with the Thr120 risk allele. DisclosuresNo relevant conflicts of interest to declare.