Platelet Binding Research Articles

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

Characterization of copy-number variants in a large cohort of patients with von Willebrand disease reveals a relationship between disrupted regions and disease type

BackgroundGenetic analysis for von Willebrand disease (VWD) commonly utilizes DNA sequencing to identify variants in the von Willebrand factor (VWF) gene; however, this technique cannot always detect copy-number variants (CNVs). Additional mapping of CNVs in patients with VWD is needed. ObjectivesThis study aimed to characterize CNVs in a large sample of VWF mutation-negative VWD patients. MethodsTo determine the role of CNVs in VWD, a VWF high-resolution comparative genomic hybridization array was custom-designed to avoid multiple sequence variations, repeated sequences, and the VWF pseudogene. This was performed on 204 mutation-negative subjects for whom clinical variables were also available. ResultsAmong the 204 patients, 7 unique CNVs were found, with a total of 24 CNVs (12%). Of the 7 unique CNVs, 1 was novel, 1 was found in a VWF database, and 5 were previously reported. All patients with type 1C VWD and a CNV had the same exon 33 and 34 in-frame deletion. Certain clinical variables were also significantly different between those with and without CNVs. ConclusionThe in-frame deletion in patients with type 1C VWD exactly matches the D4N module of the D4 domain, a region where mutations and deletions are known to affect clearance. We observed significantly higher VWF–to–ristocetin cofactor levels in patients with type 1C VWD and a CNV than in patients without a CNV, suggesting a relationship between CNVs and the increased clearance observed in patients with type 1C VWD. Glycoprotein IbM activity was significantly lower in patients with type 1 VWD and a CNV than in patients without a CNV, suggesting that platelet binding is more affected by CNVs than single base pair mutations. This work elucidates some of the underlying genetic mechanisms of CNVs in these patients.

Effect of far-infrared radiation therapy on von Willebrand factor in patients with chronic kidney disease.

Hemostatic abnormality has contributed to vascular access thrombosis in patients with chronic kidney disease (CKD). Previous studies have demonstrated that far-infrared radiation (FIR) therapy can maintain the patency and maturity of arteriovenous fistulas of patients undergoing hemodialysis (HD). However, prolonged access bleeding is observed once FIR is conducted at the end of dialysis. FIR can block the binding of platelet and von Willebrand factor (vWF), a predictor of hemostatic abnormality and vascular access thrombosis. However, clinical studies exploring FIR and vWF are sparse. We recruited 20 HD patients, 21 CKD patients, and 20 controls to examine the alteration of vWF and a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) following a single 40-min session of FIR therapy. In addition, the alteration of these factors in the HD group was examined following a 40-min FIR session thrice a week for 3 months. A decreasing trend in the vWF activity-antigen ratio of participants in all groups following a single FIR session was observed. In addition, the ratio in the HD group was significantly lower following 3 months of FIR therapy. The subgroup analysis revealed a consistent trend and multiple regression analysis showed that participants not taking hydroxymethylglutaryl-coenzyme A reductase inhibitor, diabetes mellitus, and higher hemoglobin levels were the significant factors. The alteration of the vWF activity-antigen ratio correlated moderately to that of ADAMTS13 antigen and activity. FIR may alter the ratio of ultra-large vWF multimers through ADAMTS13, contributing to inhibiting platelet-endothelium interactions of CKD patients.

Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet-neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet-HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Platelet factor XIII-A regulates platelet function and promotes clot retraction and stability

BackgroundFactor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α2-antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied. ObjectivesThis study aims to identify the role of platelet FXIII-A in platelet function. MethodsWe used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests. ResultsOur data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent. ConclusionOverall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles.

Dual antiplatelet therapy inhibits neutrophil extracellular traps to reduce liver micrometastases of intrahepatic cholangiocarcinoma

The involvement of neutrophil extracellular traps (NETs) in cancer metastasis is being clarified, but the relationship between intrahepatic cholangiocarcinoma (iCCA) and NETs remains unclear. The presence of NETs was verified by multiple fluorescence staining in clinically resected specimens of iCCA. Human neutrophils were co-cultured with iCCA cells to observe NET induction and changes in cellular characteristics. Binding of platelets to iCCA cells and its mechanism were also examined, and their effects on NETs were analyzed in vitro and in in vivo mouse models. NETs were present in the tumor periphery of resected iCCAs. NETs promoted the motility and migration ability of iCCA cells in vitro. Although iCCA cells alone had a weak NET-inducing ability, the binding of platelets to iCCA cells via P-selectin promoted NET induction. Based on these results, antiplatelet drugs were applied to these cocultures in vitro and inhibited the binding of platelets to iCCA cells and the induction of NETs. Fluorescently labeled iCCA cells were injected into the spleen of mice, resulting in the formation of liver micrometastases coexisting with platelets and NETs. These mice were treated with dual antiplatelet therapy (DAPT) consisting of aspirin and ticagrelor, which dramatically reduced micrometastases. These results suggest that potent antiplatelet therapy prevents micrometastases of iCCA cells by inhibiting platelet activation and NET production, and it may contribute to a novel therapeutic strategy.

Enhanced Thromboresistance and Endothelialization of a Novel Fluoropolymer-Coated Left Atrial Appendage Closure Device

BackgroundDevice-related thrombus (DRT) after left atrial appendage closure (LAAC) procedures is a rare but potentially serious event. Thrombogenicity and delayed endothelialization play a role in the development of DRT. Fluorinated polymers are known to have thromboresistant properties that may favorably modulate the healing response to an LAAC device. ObjectivesThe goal of this study was to compare the thrombogenicity and endothelial coverage (EC) after LAAC between the conventional uncoated WATCHMAN FLX (WM) and a novel fluoropolymer-coated WATCHMAN FLX (FP-WM). MethodsCanines were randomized for implantation with WM or FP-WM devices and given no postimplant antithrombotic/antiplatelet agents. The presence of DRT was monitored by using transesophageal echocardiography and verified histologically. The biochemical mechanisms associated with coating were assessed by using flow loop experiments to quantify albumin adsorption, platelet adhesion, and porcine implants to quantify EC and the expression of markers of endothelial maturation (ie, vascular endothelial-cadherin/p120-catenin). ResultsCanines implanted with FP-WM exhibited significantly less DRT at 45 days than those implanted with WM (0% vs 50%; P < 0.05). In vitro experiments showed significantly greater albumin adsorption (52.8 [IQR: 41.0-58.3] mm2 vs 20.6 [IQR: 17.2-26.6] mm2; P = 0.03) and significantly less platelet adhesion (44.7% [IQR: 27.2%-60.2%] vs 60.9% [IQR: 39.9%-70.1%]; P < 0.01) on FP-WM. Porcine implants showed significantly greater EC by scanning electron microscopy (87.7% [IQR: 83.4%-92.3%] vs 68.2% [IQR: 47.6%-72.8%]; P = 0.03), and higher vascular endothelial-cadherin/p120-catenin expression after 3 months on FP-WM compared with WM. ConclusionsThe FP-WM device showed significantly less thrombus and reduced inflammation in a challenging canine model. Mechanistic studies indicated that the fluoropolymer-coated device binds more albumin, leading to reduced platelet binding, less inflammation, and greater EC.

Atorvastatin-mediated inhibition of prenylation of Rab27b and Rap1a in platelets attenuates their prothrombotic capacity and modulates clot structure

Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

BackgroundThe von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. ObjectiveTo determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. MethodsWe combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. ResultsWe show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. ConclusionsOur data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

Sialofucosylation Enables Platelet Binding to Myeloma Cells via P-Selectin and Suppresses NK Cell-Mediated Cytotoxicity

Multiple myeloma (MM) is a plasma cell disorder that develops in the bone marrow (BM) and is characterized by uncontrolled proliferation and the ability to disseminate to different sites of the skeleton. Sialofucosylated structures, particularly Sialyl Lewis a/x (SLea/x), facilitate the homing of MM cells into the BM, leading to resistance to bortezomib in vivo. Platelets have been shown to play an important role in tumor metastasis. Platelets can bind to the surface of cancer cells, forming a "cloak" that protects them from the shear stress of the bloodstream and natural killer (NK) cell-mediated cytotoxicity. In this study, we showed that the presence of SLea/x induced a strong binding of MM cells to P-selectin, leading to specific and direct interactions with platelets, which could be inhibited by a P-selectin-blocking antibody. Importantly, platelets surrounded SLea/x-enriched MM cells, protecting them from NK cell-mediated cytotoxicity. The interactions between the platelets and MM cells were also detected in BM samples obtained from MM patients. Platelet binding to SLea/x-enriched MM cells was increased in patients with symptomatic disease and at relapse. These data suggest an important role of SLea/x and platelets in MM disease progression and resistance to therapy.

Abstract 3940: Validation of TFX1, a first-in-class target for cancer metastasis

Abstract Most of the drugs approved for metastatic epithelial carcinomas treat the proliferation of the tumor and not the biology of metastasis. To date, all clinical trials around metastasis that focused on proteases, kinases, or integrins have failed in clinics. As a result, metastasis biology-focused drug discovery has been deprioritized.We asked the question as to what the rate-limiting steps of metastasis were. To answer it, we created a cell-based functional assay platform, METAssay™, that dissected all metastasis biology steps into thirty cellular assays and characterization steps. The platform identified the functional differences between metastatic and proliferating cells. This platform was normalized with patient tumor cells, and a machine learning algorithm, METSCAN™, was subsequently applied to identify the weighted steps that contributed towards successful metastasis. This led to the identification of four first-in-class targets; two transcription factors, one cytokine receptor and one nuclear hormone receptor.We then validated the relevance of the first target, TFX1, by overexpressing it in both colorectal and triple-negative breast cancer cell lines. Interestingly, apart from the expected increase in migration and invasion, all cell lines showed an increase in ROS, Autophagy, PDL-1, CD-73, quiescence, exosome uptake and a decrease in apoptosis, mitosis, and exosome secretion. Conversely, the downregulation of TFX1 showed the opposite effect. TFX1 was purified, and a biochemical isothermal calorimetry (ITC) assay was standardized with a tool compound with a KD of 107 nM. The compound effectively reduced metastatic properties in the cell lines and in multiple patient samples, as analyzed by the METAssay™ platform. Colorectal cancer cell lines overexpressing TFX1 also showed increased platelet binding, which was reduced by the tool compound, but not triple-negative cancer cell lines. Also, the effect of overexpression in non-metastatic cells was more profound than in metastatic cells, suggesting a saturating effect. Further characterization of the tool compound is currently ongoing to help establish a baseline for a novel discovery programme.The ideal drug to delay metastasis should be given to primary tumor patients with no evidence of clinical metastasis, irrespective of their pathological grading. Therefore, it should have a high bar for safety. Our goal is to collaborate on a discovery journey to identify candidates and create a companion diagnostic that will identify primary tumor patients at high risk of metastasis, thereby selecting the right patient cohort for clinical trials. Citation Format: Arnab Roy Chowdhury, Manoj Pandre, Chinmaya Amarkanth, Debabani Roy Chowdhury, John Ellingboe. Validation of TFX1, a first-in-class target for cancer metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3940.

Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbβ3 integrin

BackgroundThe circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability. MethodsIn vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. ResultsUnder flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. ConclusionsOur results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control.

Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMECD), or from nominally healthy donors (controls; HCMECC). Using fluorescence microscopy, WPBs in HCMECC (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMECD (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMECD revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMECD WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMECD were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

Stimulation of platelet P2Y1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling.

Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP. Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs. 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.

Frontiers in pathophysiology and management of thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a fatal disease in which platelet-rich microthrombi cause end-organ ischemia and damage. TTP is caused by markedly reduced ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. Hereditary or congenital TTP (cTTP) is caused by ADAMTS13 gene mutations. In acquired or immune TTP (iTTP), ADAMTS13 activity is reduced by anti-ADAMTS13 autoantibodies. TTP is characterized by thrombocytopenia, hemolytic anemia, fever, renal dysfunction, and neuropsychiatric symptoms. Therapeutic plasma exchange (TPE) and immunosuppressive therapy are the mainstays of treatment. As untreated TTP has a high mortality rate, immediate initiation of TPE is recommended when TTP is suspected. Conventionally, corticosteroids have been used for immunosuppressive therapy. Current drug therapies include rituximab, an anti-CD20 antibody that is effective in newly diagnosed cases and refractory cases, as well as for relapse prevention, and caplacizumab, an anti- von Willebrand factor (VWF) nanobody that inhibits the binding of platelets to VWF and prevents microthrombi formation. Recombinant human ADAMTS13 is a promising treatment for cTTP. Although these therapeutic advances have improved the outcomes of TTP, early diagnosis and prompt initiation of appropriate therapy are necessary to achieve these outcomes.

Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2V617F mutation

BackgroundJAK2V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis. ObjectivesOur main objective was to explore and compare platelet function depending on the MPN molecular marker. MethodsWe analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels. ResultsWe observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN. ConclusionsThese results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR.

Platelet count, temperature and pH value differentially affect hemostatic and immunomodulatory functions of platelets

Platelets are primarily recognized for their role in hemostasis, but also regulate immune responses by interacting with leukocytes. Their highly sensitive nature enables platelets to rapidly respond to micro-environmental changes, which is crucial under physiological condition but can jeopardize in vitro analyses. Thus, we tested how platelet count and changes in pH and temperatures, which are commonly experienced during inflammation and infection but also affected by ex vivo analyses, influence platelet-leukocyte interaction and immunomodulation.Reducing platelet count by up to 90 % slightly decreased platelet activation and platelet-leukocyte aggregate formation, but did not affect CD11b activation nor CD62L shedding of monocytes or neutrophils. Acidosis (pH 6.9) slightly elevated platelet degranulation and binding to innate leukocytes, though pH changes did not modulate leukocyte activation. While platelet responsiveness was higher at room temperature than at 37 °C, incubation temperature did not affect platelet-leukocyte aggregate formation. In contrast, platelet-mediated CD11b activation and CD62L expression increased with temperature.Our data thus demonstrate the importance of standardized protocols for sample preparation and assay procedure to obtain comparable data. Further, unspecific physiologic responses such as thrombocytopenia, acidosis or temperature changes may contribute to platelet dysfunction and altered platelet-mediated immunomodulation in inflammatory and infectious disease.

An Updated Review on Glycoprotein IIb/IIIa Inhibitors as Antiplatelet Agents: Basic and Clinical Perspectives.

The glycoprotein (GP) IIb/IIIa receptor is found integrin present in platelet aggregations. GP IIb/IIIa antagonists interfere with platelet cross-linking and platelet-derived thrombus formation through the competition with fibrinogen and von Willebrand factor. Currently, three parenteral GP IIb/IIIa competitors (tirofiban, eptifibatide, and abciximab) are approved for clinical use in patients affected by percutaneous coronary interventions (PCI) in the location of acute coronary syndrome (ACS). GP IIb/IIIa antagonists have their mechanism of action in platelet aggregation prevention, distal thromboembolism, and thrombus formation, whereas the initial platelet binding to damage vascular areas is preserved. This work is aimed to provide a comprehensive review of the significance of GP IIb/IIIa inhibitors as a sort of antiplatelet agent. Their mechanism of action is based on factors that affect their efficacy. On the other hand, drugs that inhibit GP IIb/IIIa already approved by the FDA were reviewed in detail. Results from major clinical trials and regulatory practices and guidelines to deal with GP IIb/IIIa inhibitors were deeply investigated. The cardiovascular pathology and neuro-interventional surgical application of GP IIb/IIIa inhibitors as a class of antiplatelet agents were developed in detail. The therapeutic risk/benefit balance of currently available GP IIb/IIa receptor antagonists is not yet well elucidated in patients with ACS who are not clinically evaluated regularly for early cardiovascular revascularization. On the other hand, in patients who have benefited from PCI, the antiplatelet therapy intensification by the addition of a GP IIb/IIIa receptor antagonist (intravenously) may be an appropriate therapeutic strategy in reducing the occurrence of risks of thrombotic complications related to the intervention. Development of GP IIb/IIIa inhibitors with oral administration has the potential to include short-term antiplatelet benefits compared with intravenous GP IIb/IIIa inhibitors for long-term secondary preventive therapy in cardiovascular disease. But studies showed that long-term oral administration of GP IIb/IIIa receptor inhibitors has been ineffective in preventing ischemic events. Paradoxically, they have been linked to a high risk of side effects by producing prothrombotic and pro-inflammatory events.

Expression of Concern: Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface.

Expression of Concern: Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface.

Pervasive Platelet Secretion Defects in Patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Critically ill COVID-19 patients suffer from thromboembolic as well as bleeding events. Endothelial dysfunction, spiking of von Willebrand factor (vWF), and excessive cytokine signaling result in coagulopathy associated with substantial activation of plasmatic clotting factors. Thrombocytopenia secondary to extensive platelet activation is a frequent finding, but abnormal platelet dysfunction may also exist in patients with normal platelet counts. In this study, we performed analyses of platelet function and of von Willebrand factor in critically ill COVID-19 patients (n = 13). Platelet aggregometry was performed using ADP, collagen, epinephrin, and ristocetin. VWF and fibrinogen binding of platelets and CD62 and CD63 expression after thrombin stimulation were analyzed via flow cytometry. In addition, VWF antigen (VWF:Ag), collagen binding capacity (VWF:CB), and multimer analysis were performed next to routine coagulation parameters. All patients exhibited reduced platelet aggregation and decreased CD62 and CD63 expression. VWF binding of platelets was reduced in 12/13 patients. VWF:CB/VWF:Ag ratios were pathologically decreased in 2/13 patients and elevated in 2/13 patients. Critically ill COVID-19 patients exhibit platelet secretion defects independent of thrombocytopenia. Platelet exhaustion and VWF dysfunction may result in impaired primary hemostasis and should be considered when treating coagulopathy in these patients.