Platelet Shape Change Research Articles

Assay variables and early clinical evaluation of low-angle light scattering for platelet function analysis.

The recently developed platelet aggregation technique based on low-angle light scattering (LaSca) in diluted platelet-rich plasma (PRP) requires only a small sample volume and provides information about platelet aggregation and shape change. This study aimed to investigate the influence of preanalytical and analytical variables and to validate the method in a real-life pediatric hematology hospital setting. Platelet aggregation was induced by ADP in diluted PRP in the presence of 2mM calcium at 23°C. The study included healthy adults (n = 30), healthy children (n = 20), and pediatric patients with suspected or diagnosed platelet function abnormalities (n = 25). The assay parameters were stable for at least 3h after isolation of PRP and were sensitive to plasma dilution in the range of 2-8%. The initial aggregation velocity was significantly reduced in pediatric patients compared with healthy children (p < 0.05). ADP-induced light transmission amplitude was moderately correlated with LaSca amplitude of aggregation in healthy children (p = 0.52, p < 0.05) but not in pediatric patients. We standardized the protocol for platelet aggregation assessment by LaSca and characterized the influence of preanalytical and analytical variables on it.

LRRC8 complexes are adenosine nucleotide release channels regulating platelet activation and arterial thrombosis.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y 1 and P2Y 12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo . These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Gelsolin controls the release of phosphatidylserine (PS)-positive microvesicles (MVs) from platelets

Upon activation by vascular injury or extracellular agonists, platelets undergo rapid change shape, a process regulated by the actin cytoskeleton and accessory proteins. Platelet shape change is accompanied by the secretion of hemostatic factors and immunomodulatory cytokines from their intracellular granules, as well as the release of microvesicles (MVs) containing pro-inflammatory cytokines and procoagulant phosphatidylserine (PS). However, the role of actin dynamics in MV generation remains unclear. In this study, we found that blocking actin polymerization with cytochalasin D attenuated the release of PS-positive MVs in human platelets stimulated by thrombin or the calcium ionophore A23187. The actin-severing protein gelsolin (Gsn) facilitates normal actin filament turnover in activated platelets. Platelets from Gsn-deficient (Gsn−/−) mice showed reduced MV release compared to platelets from control mice. These findings indicate that the proper dynamics of the actin cytoskeleton are essential for MV generation in platelets, which has implications for their pro-inflammatory and procoagulant functions.

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC). The NO-GC/cGMP pathway in platelets has been extensively studied. However, its role in regulating the biomechanical properties of platelets has not yet been addressed and remains unknown. We therefore investigated the stiffness of living platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat using scanning ion conductance microscopy (SICM). Stimulation of human and murine platelets with cGMP-modulating drugs decreased cellular stiffness and downregulated P-selectin, a marker for platelet activation. We also quantified changes in platelet shape using deep learning-based platelet morphometry, finding that platelets become more circular upon treatment with cGMP-modulating drugs. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulfate (ABC)-containing regimens. Our results corroborate a functional role of the NO-GC/cGMP pathway in platelet biomechanics, indicating that biomechanical properties such as stiffness or shape could be used as novel biomarkers in clinical research.

Antiplatelet and Antithrombotic Activities of Lespedeza cuneata via Pharmacological Inhibition of Integrin αIIbβ3, MAPK, and PI3K/AKT Pathways and FeCl3-Induced Murine Thrombosis.

Cardiovascular diseases (CVDs) have been the major cause of mortality all around the globe. Lespedeza cuneata abbreviated as L. cuneata with the authority name of Dumont de Courset (G. Don) is a perennial flowering plant commonly grown in Asian countries such as Korea, Japan, China, and Taiwan. We aimed to investigate the L. cuneata extract's antiplatelet and antithrombotic properties as GC-MS analysis indicated that the extract contained short-chain fatty acids, which have been reported to possess beneficial cardiovascular effects. L. cuneata was extracted using water, 50% EtOH, 70% EtOH, and 100% EtOH. For in vitro antiplatelet analysis, washed platelets were prepared and incubated with L. cuneata with 200 μg/mL of 50% EtOH in the presence of 1 mM of CaCl2 for 1 minute followed by agonist (collagen 2.5 μg/mL or ADP 10 μM or thrombin 0.1 U/mL) stimulation for 5 minutes over light transmission aggregometer. Scanning electron microscopy was performed to assess platelet shape change. ATP release and intracellular calcium mobilization were quantified to assess the granular content. Fibrinogen-binding assay and clot retraction assay assessed integrin αIIbβ3-mediated inside-out and outside-in signaling. Protein phosphorylation expression was investigated by western blot analysis. Finally, the in vivo antithrombotic efficacy was investigated by oral dosage of L. cuneata 200 and 400 mg/kg and aspirin 100 mg/kg for 7 days, and tail bleeding and FeCl3-induced murine thrombus model were performed. In vitro platelet aggregation and platelet shape change were dose-dependently suppressed by L. cuneata. Calcium mobilization, dense granules secretion, integrin αIIbβ3-mediated inside-out and outside-in signaling, and protein phosphorylation of MAPK and PI3K/Akt pathways were significantly inhibited. In vivo assays revealed that L. cuneata prevents side effects of synthetic drugs via nonsignificantly increasing bleeding time and improving coronary artery blood flow and animal survival. Our results demonstrate that L. cuneata exhibited potent antiplatelet and antithrombotic effects and can be considered a potential herbal medicine with cardioprotective effects.

Investigation of platelet activation and calcium store release by a topical hemostatic xerogel dressing for improved blood clotting

AbstractAdequate topical hemostatic dressings are required as first‐line approach for reduction of uncontrolled hemorrhage, a leading cause of mortalities. Platelets play a major role during blood clotting to prevent hemorrhage during injuries. Here, we demonstrate the mechanisms activated through protease activating receptor 1 (PAR1), a platelet membrane protein in response to a porous composite xerogel dressing incorporating SiNPs (size 122 ± 10 nm) and calcium (2.5 mM), characterized by scanning electron microscopy–energy dispersive x‐ray spectroscopy and Fourier transform infrared spectroscopy. The composite displayed 13.9‐fold improved blood clotting index in comparison to commercial dressing. The composite displayed increased platelet aggregation due to development of well‐formed pseudopodia as compared to bare xerogel, SFLLRN (a thrombin mimic), adenosine di‐phosphate (a platelet activator), and heparin (a thrombin inhibitor). Further, PAR1 gene was significantly upregulated in model A549 epithelial cell line (1.2‐fold) and human platelets (1.4‐fold). The composite enhanced calcium release and its extrusion in A549 cells. Upregulation of PAR1 on the platelet surface and calcium release are crucial for platelet shape change and aggregation. The data indicate that xerogel composite containing SiNPs and calcium enhanced blood clotting through activation of PAR1. Such dressings can provide a potential hemostatic solution to reduce blood loss, disability, and mortality during surgery and trauma care.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

The role of platelet P2Y12 receptors in inflammation.

Inflammation is a complex pathophysiological process underlying many clinical conditions. Platelets contribute to the thrombo-inflammatory response. Platelet P2Y12 receptors amplify platelet activation, potentiating platelet aggregation, degranulation and shape change. The contents of platelet alpha granules, in particular, act directly on leucocytes, including mediating platelet-leucocyte aggregation and activation via platelet P-selectin. Much evidence for the role of platelet P2Y12 receptors in inflammation comes from studies using antagonists of these receptors, such as the thienopyridines clopidogrel and prasugrel, and the cyclopentyltriazolopyrimidine ticagrelor, in animal and human experimental models. These suggest that antagonism of P2Y12 receptors decreases markers of inflammation with some evidence that this reduces incidence of adverse clinical sequelae during inflammatory conditions. Interpretation is complicated by pleiotropic effects such as those of the thienopyridines on circulating leucocyte numbers and of ticagrelor on adenosine reuptake. The available evidence suggests that P2Y12 receptors are prominent mediators of inflammation and P2Y12 receptor antagonism as a potentially powerful strategy in a broad range of inflammatory conditions. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

PB0424 The Critical Role of Filamin a in the Platelet Shape Change Reaction

PB0424 The Critical Role of Filamin a in the Platelet Shape Change Reaction

Dematin Regulates Calcium Mobilization, Thrombosis, and Early Akt Activation in Platelets.

The complex intrinsic and extrinsic pathways contributing to platelet activation profoundly impact hemostasis and thrombosis. Detailed cellular mechanisms that regulate calcium mobilization, Akt activation, and integrin signaling in platelets remain incompletely understood. Dematin is a broadly expressed actin binding and bundling cytoskeletal adaptor protein regulated by phosphorylation via cAMP-dependent protein kinase. Here, we report the development of a conditional mouse model specifically lacking dematin in platelets. Using the new mouse model termed PDKO, we provide direct evidence that dematin is a major regulator of calcium mobilization, and its genetic deletion inhibits the early phase of Akt activation in response to collagen and thrombin agonists in platelets. The aberrant platelet shape change, clot retraction, and invivo thrombosis observed in PDKO mice will enable future characterization of dematin-mediated integrin activation mechanisms in thrombogenic as well as nonvascular pathologies.

Rho GTPase Signaling in Platelet Regulation and Implication for Antiplatelet Therapies.

Platelets play a vital role in regulating hemostasis and thrombosis. Rho GTPases are well known as molecular switches that control various cellular functions via a balanced GTP-binding/GTP-hydrolysis cycle and signaling cascade through downstream effectors. In platelets, Rho GTPases function as critical regulators by mediating signal transduction that drives platelet activation and aggregation. Mostly by gene targeting and pharmacological inhibition approaches, Rho GTPase family members RhoA, Rac1, and Cdc42 have been shown to be indispensable in regulating the actin cytoskeleton dynamics in platelets, affecting platelet shape change, spreading, secretion, and aggregation, leading to thrombus formation. Additionally, studies of Rho GTPase function using platelets as a non-transformed model due to their anucleated nature have revealed valuable information on cell signaling principles. This review provides an updated summary of recent advances in Rho GTPase signaling in platelet regulation. We also highlight pharmacological approaches that effectively inhibited platelet activation to explore their possible development into future antiplatelet therapies.

The relative importance of platelet integrins in hemostasis, thrombosis and beyond.

Integrins are heterodimeric transmembrane receptors composed of α and β chains, with an N-terminal extracellular domain forming a globular head corresponding to the ligand binding site. Integrins regulate various cellular functions including adhesion, migration, proliferation, spreading and apoptosis. On platelets, integrins play a central role in adhesion and aggregation on subendothelial matrix proteins of the vascular wall, thereby ensuring hemostasis. Platelet integrins belong either to the β1 family (α2β1, α5β1 and α6β1) or to the β3 family (αIIbβ3 and αvβ3). On resting platelets, integrins can engage their ligands when the latter are immobilized but not in their soluble form. The effects of various agonists promote an inside-out signal in platelets, increasing the affinity of integrins for their ligands and conveying a modest signal reinforcing platelet activation, called outside-in signaling. This outside-in signal ensures platelet adhesion, shape change, granule secretion and aggregation. In this review, we examine the role of each platelet integrin in hemostatic plug formation, hemostasis and arterial thrombosis and also beyond these classical functions, notably in tumor metastasis and sepsis.

Platelet Hemostasis Reactions at Different Temperatures Correlate with Intracellular Calcium Concentration.

Hypo- and hyperthermia affect both primary and secondary hemostasis; however, there are controversial data concerning platelet activation and the underlying mechanisms under hypo- and hyperthermia. The discrepancies in the data could be partly explained by different approaches to hemostatic reactions analysis. We applied a new LaSca-TMF laser particle analyzer for a simultaneous fluorescence and laser scattering analysis of platelet responses at different temperatures. Human platelets were activated by ADP in a wide range of temperatures, and platelet transformations (e.g., a shape change reaction, aggregation and clot formation) and the intracellular calcium concentration ([Ca2+]i) were analyzed by LaSca-TMF and confocal microscopy. The platelet shape change reaction gradually increased with a rising temperature. The platelet aggregation strongly decreased at low ADP concentrations with the augmentation of the temperature and was independent of the temperature at high ADP concentrations. In contrast, the clotting time decreased with a temperature increase. Similar to the aggregation response, a rise in [Ca2+]i triggered by low ADP concentrations was higher under hypothermic conditions and the differences were independent of the temperature at high ADP concentrations. We showed that the key reactions of cellular hemostasis are differentially regulated by temperature and demonstrated for the first time that an accelerated aggregation under hypothermic conditions directly correlated with an increased level in [Ca2+]i in platelets.

Dual antiplatelet therapy is associated with high alpha-tubulin acetylation in platelets from coronary artery disease patients

Platelet inhibition is the main treatment strategy to prevent atherothrombosis. Despite dual antiplatelet therapy (DAPT) combining aspirin and a P2Y12 receptor inhibitor, high platelet reactivity persists in some patients due to poor response to treatment and is associated with ischemic risk. It remains unknown if circulating platelets in high-risk patients have different morphological characteristics which could participate in their pro-thrombotic potential. We postulated that alpha-tubulin acetylation, a cytoskeletal modification known to regulate platelet shape change, could reflect circulating platelet reactivity and impact their morphology. We collected arterial blood samples from 187 patients admitted for coronary angiography. Platelet reactivity was assessed in whole blood using multiplate analysis. Platelets were then isolated to evaluate alpha-tubulin acetylation level by western blotting. 141 patients were taking aspirin among which 32 were treated with an additional P2Y12 inhibitor. DAPT didn’t achieve sufficient platelet inhibition in 8 out of 32 patients. Participants provided written informed consent and the study was approved by the institutional ethics committee. Platelet alpha-tubulin acetylation was significantly increased in DAPT-treated patients ( P < 0.001). A minority of non-treated patients (11.4%) exhibited high alpha-tubulin acetylation (third acetyl alpha-tubulin tertile) whereas a high level was observed in most patients on efficient DAPT (83.3%). Interestingly, the proportion of patients with high acetyl alpha-tubulin (37.5%) level was drastically decreased in clopidogrel low responders. Multivariate logistic regression further supported that efficient DAPT is independently associated with a 27-fold increase in the likelihood of being in the highest acetyl alpha-tubulin tertile ( P < 0.001). We revealed high platelet alpha-tubulin acetylation as a potential marker of efficient platelet inhibition by dual antiplatelet therapy. Since alpha-tubulin acetylation is a hallmark of stable microtubules, its increase could contribute to maintaining resting morphology of circulating platelets.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Platelets and Antiplatelet Medication in COVID-19-Related Thrombotic Complications.

Coronavirus disease 2019 (COVID-19) induces a hypercoagulatory state that frequently leads to thromboembolic complications. Whereas anticoagulation is associated with reduced mortality, the role of antiplatelet therapy in COVID-19 is less clear. We retrospectively analyzed the effect of anticoagulation and antiplatelet therapy in 578 hospitalized patients with COVID-19 and prospectively monitored 110 patients for circulating microthrombi and plasma markers of coagulation in the first week of admission. Moreover, we determined platelet shape change and also thrombi in postmortem lung biopsies in a subset of patients with COVID-19. We observed no association of antiplatelet therapy with COVID-19 survival. Adverse outcome in COVID-19 was associated with increased activation of the coagulation cascade, whereas circulating microthrombi did not increase in aggravated disease. This was in line with analysis of postmortem lung biopsies of patients with COVID-19, which revealed generally fibrin(ogen)-rich and platelet-low thrombi. Platelet spreading was normal in severe COVID-19 cases; however, plasma from patients with COVID-19 mediated an outcome-dependent inhibitory effect on naïve platelets. Antiplatelet medication disproportionally exacerbated this platelet impairment in plasma of patients with fatal outcome. Taken together, this study shows that unfavorable outcome in COVID-19 is associated with a profound dysregulation of the coagulation system, whereas the contribution of platelets to thrombotic complications is less clear. Adverse outcome may be associated with impaired platelet function or platelet exhaustion. In line, antiplatelet therapy was not associated with beneficial outcome.