ABSTRACT:Inherited genetic variants that modulate platelet function contribute significantly to thrombotic disorders, yet their mechanisms and clinical implications remain underexplored. Two genome-wide association studies identified an A→G variant (rs10886430) in the first intron of G protein-coupled receptor kinase 5 (GRK5), found in homozygosity in ∼5 million Americans. The homozygous GRK5 GG genotype is associated with an increased risk of stroke and venous thromboembolism, but the mechanistic link between this variant and thrombotic risk has remained unclear. To investigate this, we identified 3 GG individuals. GRK5 protein levels in GG platelets were 90% lower than in AA controls. The significant reduction in GRK5 levels in GG platelets led to elevated platelet responsiveness to thrombin and a protease-activated receptor 1 (PAR1) agonist but not a PAR4 agonist. These findings were corroborated in GRK5-/- induced pluripotent stem cell-derived megakaryocytes, transgenic Grk5-deficient murine platelets, and AA platelets exposed to a GRK5 inhibitor. We demonstrated that PAR1 internalization was reduced in GG platelets, leading to enhanced PAR1 signaling. Under venous shear in an endothelialized microfluidic system, GG platelets exhibited increased accumulation, which was reversed by PAR1 inhibition with vorapaxar. In an arterial murine thrombosis model following human platelet infusion, GG platelets also showed enhanced thrombus formation in vivo. This study provides, to our knowledge, the first experimental evidence directly linking a highly prevalent human GRK5 variant to defective PAR1 regulation and increased thrombotic risk. Together, these findings establish that the GRK5 GG genotype confers increased thrombotic potential through impaired PAR1 desensitization, providing mechanistic insight that connects human genetics, thrombin receptor signaling, and thrombotic disease.

Heparin-induced thrombocytopenia (HIT) is an immune prothrombotic disorder characterized by the binding of platelet-activating immunoglobulin G antibodies to platelet factor 4/heparin. In platelets, this leads to cross-linking of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor FcγRIIa, platelet activation, and thrombocytopenia, which in combination with extensive thrombin generation significantly increases the risk of thrombosis. Our laboratory has previously demonstrated that 12-lipoxygenase (12-LOX), an oxygenase primarily expressed in platelets, plays a critical role in platelet activation through FcγRIIa. In this study, we aimed to determine the effectiveness of VLX-1005, a potent and selective inhibitor of 12-LOX, alone or in combination with argatroban, in preventing HIT. Pretreatment with VLX-1005 attenuated aggregation of human washed platelets stimulated with a HIT immune complex in vitro. VLX-1005 prevented ITAM-induced human whole-blood impedance alone or in combination with argatroban. VLX-1005 treatment impaired platelet adhesion and accumulation on a collagen-coated surface under shear stress. Mice expressing transgenic human FcγRIIa and 12-LOX experienced severe thrombocytopenia and thrombosis after a HIT-like challenge, whereas mice expressing transgenic human FcγRIIa with 12-LOX knockout were completely refractory to HIT pathology. VLX-1005 treatment did not affect coagulation or increase the risk of bleeding. This study demonstrates that inhibition of 12-LOX might be an effective intervention for preventing ITAM-regulated platelet activation, such as in HIT, and is independent of argatroban effects in blood. Importantly, VLX-1005 prevents platelet activation and does not increase the bleeding risk associated with direct thrombin inhibitors, such as argatroban.

ABSTRACT:Ehlers-Danlos syndrome (EDS) is a group of connective tissue disorders characterized by joint hypermobility, skin hyperelasticity, perivascular tissue fragility, easy bruising, and increased bleeding risk. Abnormal bleeding in EDS ranges from mild ecchymoses to life-threatening hemorrhage. Platelet function abnormalities have been reported in people with EDS, but the broad nature and extent of these defects remain poorly defined. Herein, we evaluated blood samples from people with the hypermobile, classical, classical-like, and vascular types of EDS, and used a Col5a1+/- mouse model of classical EDS to characterize the extent of platelet dysfunction. Our findings suggest that platelet dysfunction in EDS is an outcome of reduced integrin αIIbβ3 activation resulting from decreased phosphorylation of talin-1, leading to defects in aggregation and spreading. The observed platelet dysfunction was associated with reduced expression of the platelet surface receptors glycoprotein VI (GPVI) and proteinase-activated receptor 1 (PAR1) and impaired downstream signaling. Col5a1+/- mice demonstrated increased tail bleeding time, reproduced the signaling defects observed in platelets from people with EDS, and exhibited decreased susceptibility to FeCl3-induced carotid artery thrombosis. Collectively, our data indicate that platelet dysfunction in EDS is likely contributing to hemorrhagic complications.

BACKGROUND:Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction to heparin caused by pathological immunoglobulin G (IgG) antibodies targeting platelet factor (PF) 4/heparin antigenic complexes. Immune complexes (ICs) consisting of IgG antibodies and PF4/heparin bind to cellular FcγRIIA receptors to initiate prothrombotic cell signaling, including cytokine production and release. OBJECTIVES:This study sought to investigate the role of inflammasome cytokine production and release of interleukin (IL)-1β following HIT IC challenge. METHODS:Inflammasome cytokine release of IL-1β was measured in a whole blood assay, where healthy donor whole blood was incubated with PF4/heparin antigen and ICs consisting of the monoclonal HIT-like antibody, KKO, or HIT patient plasma. Corresponding studies were conducted in peripheral blood mononuclear cells (PBMCs). Requirements for cellular FcγRIIA, complement pathway, and inflammasome were determined with respective inhibitors. FcγRIIA transgenic mice with or without a genetic deletion of Nlrp3 were subjected to an FcγRIIA-dependent thrombosis model. RESULTS:Whole blood or PBMCs incubated with HIT ICs or HIT patient plasma elicited a significant increase in IL-1β secretion, which was dependent on FcγRIIA. IL-1β secretion required complement, as heat-inactivated plasma or incubation with terminal complement inhibitors prevented cytokine release. Pretreatment of whole blood or PBMCs with the NLRP3 inhibitor MCC950 markedly reduced IL-1β secretion. Furthermore, transgenic mice given an FcγRIIA-dependent thrombotic challenge demonstrated a requirement for Nlrp3 to experience severe thrombocytopenia and thrombosis. CONCLUSION:These data demonstrate that HIT ICs activate the inflammasome pathway in an FcγR/complement-dependent manner leading to IL-1β secretion, which is a positive regulator of HIT pathology.

Low-dose aspirin is recommended for prevention of hypertensive disorders of pregnancy (HDP) and preterm birth (PTB) in high-risk pregnancies. There is limited data on factors impacting aspirin response in pregnancy. We aimed to evaluate predictors of aspirin response and association with pregnancy outcome with a prospective study of high-risk pregnancies taking 81 mg aspirin daily. Aspirin response was evaluated with Platelet Function Assay-100 (PFA-100) epinephrine closure time at baseline (< 16 weeks' gestation), follow-up 1 (2-4 weeks after aspirin initiation), and follow-up 2 (28-32 weeks gestation). Multivariable regression was used to identify factors associated with PFA-100 at each visit, and results presented with beta coefficient (B) and confidence interval. The median difference (MD) in PFA-100 in those with and without HDP or PTB was compared. Results included 108 who completed follow-up 1 and 96 who completed both visits with > 75% adherence. PFA-100 was increased from baseline at follow-ups 1 and 2 (MD 37 (27-49); MD 26 (15.5-38.5) respectively). At follow-up 1, obesity (B = -30 (-53 to -7) seconds), diabetes (B = -39 (-75 to -2) seconds), and age (B = 2.2 (0.3-4.0) seconds per year increased) were associated with PFA-100 response. Those with HDP in the current pregnancy versus not had similar aspirin response, but those with PTB versus term birth in the current pregnancy had reduced aspirin response at 28-32 weeks (MD -27 (-54 to -3) seconds). A daily dose of 81 mg aspirin results in platelet inhibition throughout gestation. Obesity, diabetes, and younger age are associated with reduced aspirin response in pregnancy.

Abstract: The platelet protease-activated receptor 4 (PAR4) threonine 120 (Thr120) allele is an activating allele associated with reduced aspirin response in vitro. Aspirin is recommended in high-risk pregnancies to prevent preeclampsia and preterm birth. We evaluated the impact of PAR4 genotype on aspirin response in pregnancy, as measured by platelet function assay 100 (PFA-100) epinephrine closure time, and perinatal outcomes. We conducted a prospective cohort study of high-risk pregnant patients who took 81-mg aspirin daily. PFA-100 was assessed at baseline, 2 to 4 weeks after aspirin initiation (follow-up 1), and 28 to 32 weeks’ gestation (follow-up 2). Primary outcome was difference in PFA-100 by genotype. Exposure was defined as PAR4-Thr120 homozygous vs not. Of the 122 participants were included, 24 (19.6%) were PAR4-Thr120 homozygous, and 106 completed follow-up 1 with >75% adherence. Participants homozygous for PAR4-Thr120 had a significantly higher rate of prior preterm birth (50.0% vs 16.1%; P = .004). Genotype was not significantly associated with PFA-100 response in multivariable regression. In the subset with urinary thromboxane data available (n = 18), thromboxane levels were higher in those who were homozygous vs not (geometric mean ratio, 208 [95% confidence interval, 1.66-2.61]; P < .001) in multivariable regression. There was a higher rate of placental intervillous thrombosis, although not statistically significant (16.7% vs 3.9%; P = .08). Patients homozygous for PAR4-Thr120 had a higher incidence of prior preterm birth, a risk factor for poor perinatal outcome. Aspirin response, measured by PFA-100, was similar across genotypes, although Thr120 homozygosity may be associated with reduced thromboxane suppression and a higher rate of placental vasculopathy even with 81-mg aspirin daily.

Aspirin is recommended in high-risk pregnancies for the prevention of preeclampsia/preterm birth; it is unclear whether aspirin's antiplatelet or anti-inflammatory (or both) effects drive its efficacy, and there are no established markers of therapeutic efficacy for pregnancy-specific outcomes. MicroRNAs (miR) are noncoding posttranscriptional modifiers implicated in preeclampsia/preterm birth and platelet function. We aimed to evaluate whether selected platelet-associated miRs were associated with platelet inhibition following aspirin use and associated with perinatal outcomes.This is a planned secondary analysis of a cohort of high-risk singleton pregnancies taking aspirin. Participants had bloodwork done in the first trimester, preaspirin, 2 to 4 weeks after aspirin initiation (follow-up 1), and at 28 to 32 weeks of gestation (follow-up 2). We selected six platelet-associated miRs (16, 18a, 126, 155, 181a, and 223) that are also associated with preeclampsia and reported quantity as fold-change from baseline (follow-up/baseline). We evaluated with univariate analysis the relationship between miR fold-change at follow-up visits 1 and 2 with Platelet Function Assay-100 epinephrine closure time (PFA-100, Siemens), a measure of aspirin response. We also evaluated the relationship between miR fold-change from baseline at each visit and the outcome of (1) hypertensive disorders of pregnancy (HDP): preeclampsia or gestational hypertension and (2) preterm birth < 37 weeks.Of the original cohort, 57 were included in this secondary analysis. The fold-change in miR-223 and miR-18a significantly decreased with increasing PFA-100 epinephrine closure time. Twelve (21%) pregnancies had HDP, and eight (14%) had preterm birth < 37 weeks. Four preterm births were due to severe preeclampsia, and one was preeclampsia without severe features and spontaneous labor. Fold-change in miRs was not associated with HDP, but increased miR-223 at 28 to 32 weeks was significantly associated with an increased risk of preterm birth (odds ratio: 2.77 [1.21-6.87]; p = 0.04).Selected platelet-associated miRs may be promising markers to assess the therapeutic effect of aspirin in pregnancy and warrant further exploration. · Platelet-associated miR decreases in association with platelet inhibition.. · Elevated miR-223 in the third trimester may be associated with preterm birth.. · Platelet-associated miR may be a marker of aspirin therapeutic effect in pregnancy..

BACKGROUND:Phosphatidylserine (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. OBJECTIVES:Test 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. METHODS:PS-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. RESULTS:DPAL 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. CONCLUSION:DPAL 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.

BACKGROUND:Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES:The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS:A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS:PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION:PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

BACKGROUND: Low dose aspirin is recommended for prevention of preeclampsia, however there is not consensus on the appropriate dose. Pregnancy specific changes have the potential to impact the pharmacology of aspirin in pregnancy, however there are very limited studies on aspirin pharmacokinetics in pregnancy and none linking pharmacokinetics (PK, drug dose and drug level) to pharmacodynamics (PD, drug dose and physiologic response) in pregnancy. As a result, we do not have a good understanding of the pharmacologic response to aspirin in pregnancy, which has important implications for clinical efficacy. We sought to describe the PK and PD of aspirin through pregnancy and to identify individual covariates that impacted aspirin PK/PD. OBJECTIVE: We sought to describe the PK and PD of aspirin through pregnancy (first and third trimester), to identify covariates that significantly impact aspirin PK and to identify the relationship between aspirin PK and PD. STUDY DESIGN: This is a prospective study of patients at high risk for preeclampsia recommended to take 81 mg aspirin daily. This study involved 3 visits as follows: (1) baseline, first trimester (10-16 weeks of gestation) 6-hour PK visit, done before initiation of aspirin; (2) follow-up 1: 2 to 4 weeks after aspirin initiation; and (3) follow-up 2: third trimester 6-hour PK visit (28-32 weeks of gestation). The following were assessed at each visit: weight or body mass index, platelet function analysis-100 (Siemens), urinary thromboxane B2, serum thromboxane B2, and plasma salicylic acid. The PK visits consisted of blood work done at baseline (predose), administration of 81 mg nonenteric coated aspirin, and then plasma blood level of salicylic acid assessed at 30 minutes and then hourly 1 to 6 hours after dose. Pearson correlation and multivariable regression were used to identify associations between parameters and identify relevant covariates. Log-adjusted values were used for regression analysis. P<.05 was considered statistically significant. RESULTS: Nineteen participants were included with first trimester data, and 16 with third trimester data. There was no statistically significant change in mean PK parameters between the first and third trimester, although there was a trend to lower peak concentration in the third than in the first trimester (P=.08). In multivariable regression, baseline obesity and current body mass index as a continuous measures were negatively associated with log-adjusted peak salicylic acid concentration (-0.28 [-0.46 to-0.11], P=.003 and-0.02 [-0.03 to-0.009], P=.001, respectively) and log-adjusted plasma salicylic acid area under the curve 0 to 6 hours postdose (-0.25 [-0.45 to 0.05], P=.02,-0.04 [-0.07 to-0.01], P=.008 respectively). There was a significant decrease in urinary thromboxane 2 to 4 weeks after aspirin initiation compared with baseline, which correlated with a concomitant increase in platelet function analysis-100 closure time. In multivariable regression model, there was a strong association between plasma salicylic acid concentration (area under the curve 0-6 hours postdose) and urinary thromboxane (B=-3.12 [-5.38 to-1.04], P=.006), and with urinary thromboxane suppression and platelet inhibition, platelet function analysis-100 (-0.23 [-0.31 to-0.14], P<.001). With progressive thromboxane suppression, platelet inhibition (platelet function analysis-100 closure time) increased. Individual comorbidities, including weight, hypertension, and pregestational diabetes (Type I or II), also impacted aspirin response. CONCLUSION: We have demonstrated the relationship between individual factors, plasma concentrations of salicylic acid, thromboxane suppression, and platelet inhibition at a single dose (81 mg) of aspirin taken through pregnancy. Our findings suggest that dose modification of aspirin in pregnancy may help to achieve the optimal response. Our results may be used to facilitate computational modeling to identify optimal dosing, taking into consideration individual factors.

Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that is caused by IgG immune complexes that activate platelets and monocytes through Fc Receptor for IgG IIA (FcγRIIA). These immune complexes also fix complement. Stimulated monocytes produce proinflammatory cytokines including inflammasome dependent IL-1β release, which has not been investigated in HIT. Aims: Platelets have been shown to support monocyte inflammasome cytokine release during inflammation, primarily in microbial infection. This study sought to investigate the role of inflammasome cytokine production in HIT pathology. Methods: We investigated the role of the NLRP3 inflammasome following immune complex (IC) challenge in healthy donor whole blood or immortalized monocyte-like cells. The contribution of inflammasome assembly and cytokine release on HIT pathology was assessed utilizing NLRP3 inhibitor MCC950 in the triple allele ‘HIT’ mouse model. Plasma IL-1β cytokine production was measured using ELISA, and complete blood counts were taken to assess thrombocytopenia. Furthermore, the contribution of complement was assessed using IC challenge of isolated primary blood mononuclear cells suspended in plasma or heat-inactivated plasma. Results: Following human whole blood challenged or isolated mononuclear cell/platelet suspensions with two distinct immune complexes (heat aggregated IgG or heparin/PF4 complexes) we observed a significant increase in inflammasome cytokine production at 22 hours. Mononuclear/platelet cell fractions challenged with heparin/PF4 complexes had a significant increase in inflammasome cytokine secretion that was reduced with pretreatment with an FcγRIIA blocking antibody, or removal of complement with heat-inactivated plasma. We observed that administration of heparin and monoclonal αPF4/heparin antibody KKO to HIT mice resulted in thrombocytopenia and significantly increased circulating IL-1β, whereas mice treated with NLRP3 inflammasome inhibitor MCC950 protected mice from a platelet drop and cytokine production in our humanized mouse model of HIT in vivo. Conclusions: These data demonstrate for the first time that HIT IC mediates the production and secretion of inflammasome cytokines, which is influenced by the presence of platelets in a FcγRIIA dependent manner as well as by complement. Furthermore, the NLRP3 inflammasome is shown in HIT model mice to be a critical component of HIT pathophysiology. These results provide a scientific rationale for exploring mechanisms that drive platelet/monocyte/complement communication and targeting the inflammasome as a possible therapeutic strategy in HIT.

Current guidelines for treating vaccine-induced immune thrombotic thrombocytopenia (VITT) recommend non-heparin anticoagulants and intravenous immunoglobulin (IVIg). However, the efficacy of these treatments remains uncertain due to case studies involving small patient numbers, confounding factors (e.g. concurrent treatments), and a lack of animal studies. A recent study proposed danaparoid and heparin as potential VITT therapies due to their ability to disrupt VITT IgG-PF4 binding. Here, we examined the effects of various anticoagulants (including unfractionated (UF) heparin, danaparoid, bivalirudin, fondaparinux, and argatroban), IVIg, and the FcγRIIa receptor-blocking antibody, IV.3. Our investigation focused on VITT IgG-PF4 binding, platelet activation, thrombocytopenia, and thrombosis. Danaparoid, at therapeutic doses, was the sole anticoagulant that reduced VITT IgG-PF4 binding, verified by affinity-purified anti-PF4 VITT IgG. While danaparoid and high-dose UF heparin (10 U/mL) inhibited platelet activation, none of the anticoagulants significantly affected thrombocytopenia in our VITT animal model, and all prolonged bleeding time. IVIg and all anticoagulants, except UF heparin, protected VITT mice from thrombosis. Direct FcγRIIa receptor inhibition with IV.3 antibody is an effective approach for managing both thrombosis and thrombocytopenia in the VITT mouse model. Our results underscore the necessity of animal model investigations to inform and better guide clinicians on treatment choices. This study provides compelling evidence for developing FcγRIIa receptor blockers to prevent thrombosis in VITT and other FcγRIIa-related inflammatory disorders.

Objective Sepsis, which is the body’s response to overwhelming infection, can lead to septic shock, characterized by thrombocytopenia, hypotension, and organ damage. Polo-like kinase 3 (Plk3) is a ubiquitously expressed serine/threonine kinase, but its exact role in immune function is unknown.Approach and Results We used Plk3−/− and WT mice to evaluate the function of Plk3 in several models of severe sepsis. We found that WT mice die within 48 hours of 100% cecal ligation and puncture (CLP), while Plk3−/− mice survive. Survival following cecal slurry (CS) injection mirrored that of CLP as recipient WT mice succumbed, while recipient Plk3−/− mice survived. Analysis of bacterial load 24 hours after CLP revealed that WT blood and peritonea were loaded with bacteria, but bacteria were virtually undetectable in the peritonea or blood of Plk3−/− mice. To determine if bacteria infiltrate the blood of Plk3−/− mice shortly after infection, we measured bacteria 1 and 3 hours after CS injection. We found a time-dependent increase in bacteria in the blood of WT mice that was not observed in Plk3−/− mice. To determine if the lack of bacteria in the blood of Plk3−/− mice is due to enhanced clearance, we injected E. coli IV into WT and Plk3−/− mice. We found 75% mortality for both WT and Plk3−/− mice within 72 hours following IV injection suggesting that survival of Plk3−/− mice following enteric infection is likely due to reduced bacteremia.Conclusion Collectively our data suggest that Plk3 supports the systemic dissemination of bacteria and subsequent sepsis following enteric infection.### Competing Interest StatementThe authors have declared no competing interest.* Plk3 : polo-like kinase 3 WT : wildtype CLP : cecal ligation and puncture CS : cecal slurry PAMP : pathogen associated molecular pattern LPS : lipopolysaccharide PRR : pattern recognition receptor TLR4 : toll-like receptor 4 GFP : green fluorescent protein IL : interleukin CIB1 : calcium- and integrin-binding protein 1 PE : phycoerythrin AST : aspartate aminotransferase ALT : alanine aminotransferase PBS : phosphate buffered saline GPIX : glycoprotein IX WBC : white blood cell LY : lymphocyte NE : neutrophil MO : monocyte Plt : platelet

Low dose aspirin is recommended for prevention of preeclampsia in high risk patients, but individual response to aspirin is highly variable and there is no marker established to measure aspirin response in pregnancy. A commercially available assay PFA-100 epinephrine closure time (PFA-100(epi)), is used to measure aspirin response in non-pregnant adults. Increased PFA-100(epi) result indicates increased platelet inhibition, and a threshold of 150 seconds has been described for aspirin response. We aimed to characterize the response to aspirin in pregnancy with use of PFA-100(epi) and identify whether it was associated with hypertensive disorders of pregnancy (HDP). This is a prospective cohort study of singletons at high risk for preeclampsia taking 81mg aspirin daily. Blood was drawn at baseline prior to aspirin (10-16 weeks’), 2-4 weeks after aspirin initiation (follow-up 1), and at 28-32 weeks’ (follow-up 2). We evaluated PFA-100(epi) across visits as well as rate of PFA-100(epi)< 150. Primary outcome was HDP. Secondary outcomes included preterm delivery for preeclampsia (preterm preE). Multivariable logistic regression was used to evaluate the relationship between PFA-100 (epi) and outcomes, adjusting for age, BMI, and preexisting hypertension or diabetes. 103 participants had >75% adherence and completed all visits. 26% were Black, 30% had hypertension, and 10% had diabetes. PFA-100(epi) increased with aspirin through gestation (Figure). Predictors of aspirin response included diabetes; patients with diabetes had reduced PFA-100(epi) (log corrected adjusted MD -0.23 (- 0.46 to -0.01)) and higher rate of PFA-100(epi)< 150 seconds (adjusted OR 4.9 (1.1-34.3)) compared to non-diabetics. In multivariable logistic regression neither PFA-100(epi) at follow up-1 nor follow up-2 were associated with risk of HDP or preterm preE (p >0.10). Low dose (81mg) aspirin therapy in pregnancy results in platelet inhibition through gestation as measured by PFA-100(epi), but PFA-100 response was not associated with the risk of hypertensive disorders of pregnancy

Background: Platelet protease activated receptor-4 (PAR4) Thr-120 allele is an activating allele associated with increased platelet reactivity and reduced response to aspirin in vivo. We previously identified this genotype to be associated with an increased odds of preterm birth and placental vascular pathology in an unselected pregnant population. Aspirin is recommended in high risk pregnancies to prevent preeclampsia and preterm birth. It is unclear whether this genotype impacts aspirin effectiveness in pregnancy. Objective: We aimed to compare response to aspirin, as measured by Platelet Function Assay (PFA-100) epinephrine closure time, and perinatal outcomes, by PAR-4 genotype in high risk pregnant singletons recommended 81mg aspirin daily Methods: This is a planned secondary analysis of a prospective cohort study of N=130 high-risk singleton gestations recommended aspirin daily. As part of the primary study, participants were enrolled in the first trimester. They had PFA-100 epinephrine closure time assessed at baseline, and again 2-4 weeks after aspirin initiation (follow up-1) and finally at 28-32 weeks gestation (follow up-2). Participants were included if they had PAR-4 genotyping completed, took 81mg aspirin daily with at least 75% adherence by pill counting, and completed requisite follow up. Primary outcome was PFA-100 epinephrine closure time at follow up-1. Secondary outcomes were PFA-100 at follow up-2, incidence of hypertensive disorders of pregnancy and preterm birth, incidence of placental intervillous thrombosis. Exposure was defined as PAR4 Thr-120 homozygous vs not. Comparison was unadjusted with Mann Whitney U or chi-square, and adjusted with multivariable regression. Results: N=122 had genotyping complete, n=24 (19.6%) were homozygous for PAR-4 Thr120. N=106 completed follow up-1 with >75% adherence. Baseline characteristics were similar except participants homozygous for PAR4 Thr-120 had a significantly higher rate of preterm birth in a prior pregnancy (50.0% vs 16.1%, p=0.004). There was no significant difference in median aspirin response at follow-up 1 in those homozygous vs not: (152.0 [80-300] vs 151[91-300] sec, p=0.90) in unadjusted or adjusted analyses. At follow-up 2 there was a non-significant reduced response: 147.3[66-300] vs 167.0 [76-300]sec, median difference -19 (-43 to 6) sec, p=0.13, and was not significant in adjusted analysis. Rates of preeclampsia and preterm birth were similar. There was a higher rate of placental intervillous thrombosis in PAR4-Thr120 homozygous, but not statistically significant (16.7% vs 3.9%, p=0.08). Conclusion: Patients homozygous for PAR-4 Thr120 had higher incidence of prior preterm birth history, a significant risk factor for poor perinatal outcome. PFA-100 epinephrine closure time as measure of response to 81mg aspirin daily was similar across genotypes, although it is possible we were underpowered to detect a difference in the setting of other known factors (obesity, diabetes). Overall perinatal outcomes were similar, although PAR-4 Thr120 homozygous genotype may be associated with higher rate of placental vascular pathology even with daily aspirin use. Further study is needed to evaluate the role of this genotype in aspirin response, dose based response, and placental vascular pathology in high risk pregnancies.

Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.

Aspirin (81mg daily) reduces the risk of preeclampsia in high-risk individuals, however, up to 30% of treated high-risk individuals still develop preeclampsia. Aspirin response in non-pregnant adults can vary widely. The objective of this study is to identify individual factors predictive of response to aspirin therapy in pregnancy. PFA-100 closure time is a measure of platelet response to epinephrine and is a quantitative measure of aspirin induced platelet inhibition. We conducted a prospective cohort study at Thomas Jefferson University Hospital of 130 patients with a singleton pregnancy at risk for preeclampsia and recommended aspirin therapy, 81mg daily initiated ≤16 weeks. PFA-100 closure time was analyzed at baseline and then 2-4 weeks after aspirin initiation. The primary outcome was PFA-100 closure time at follow-up #1. Predictors were evaluated with multivariable least squares model with stepwise variable selection using R package. 114 participants completed follow-up visit #1 with complete data for all variables. Average gestational age at baseline was 12.6±1.5 and at follow-up#1 was 16.0 ±2.3 weeks' (p< 0.001). There was a significant increase in mean PFA-100 from baseline to follow-up #1 (131±39 vs 170±61, p< 0.001). In multivariable logistic regression, obesity and pre-gestational diabetes were associated with reduced aspirin response (lower PFA-100). Those with diabetes or obesity had lower mean PFA-100 at follow up in unadjusted and adjusted analyses (Figure). In contrast, Black race was associated with increased platelet response to aspirin therapy (MD 29 (95% CI 7-51), p=0.01) (Table). Baseline PFA-100 was also positively associated with follow-up PFA-100 (Table). Individuals with pre-gestational diabetes or obesity have a reduced platelet response to aspirin therapy. This is consistent with findings in non-pregnant adults. Aspirin dosing in pregnancy may need to be adjusted based on individual characteristics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.