Longitudinal assessment of coagulation potential before, during, and following an in vitro fertilization cycle.

INTRODUCTION: The association between estrogen and hypercoagulability is well-established but little is known about coagulation dynamics during IVF. Our goal was to measure coagulation potential prior to, during, and following an IVF cycle and to investigate differences by conception outcome. MATERIALS AND METHODS: Patients undergoing IVF with fresh embryo transfer at a single academic center using oral contraceptive pills for cycle batching underwent evaluation of thrombin generation using the calibrated automated thrombogram at multiple points during the IVF cycle. Multiple thrombin generation parameters were compared across timepoints and by IVF cycle outcome using ANOVA repeated measures analysis. RESULTS: Of the 17 patients included, 11 conceived. There was a significant increase in peak and total thrombin generation in the entire cohort between the pre-treatment natural follicular phase and following a short course of oral contraceptive pills used for cycle batching. Further increase in these parameters was seen at the time of oocyte retrieval. In the pre-treatment natural follicular phase, patients who conceived had lower peak thrombin generation. There were changes throughout the cycle for factors II, V, VIII, X, XI, XII, antithrombin, and tissue factor pathway inhibitor. Only Factor XI was distinguishable by conception status; values were lower at all visits in patients who conceived. CONCLUSION: Increases in coagulation potential are seen in patients undergoing IVF following a short course of oral contraceptive pills for cycle batching and continue during controlled ovarian hyperstimulation. Those who conceived were seen to have lower peak thrombin generation in the pre-treatment natural follicular phase.

Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Antiplatelet Effects of Flavonoid Aglycones Are Mediated by Activation of Cyclic Nucleotide-Dependent Protein Kinases.

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors.

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3-5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? AIM OF THE STUDY: In this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. MATERIALS AND METHODS: In this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). RESULTS: Validation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. CONCLUSION: Only one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Exploring the effects of Factor Xa inhibitors on thrombin generation in people with haemophilia.

Optimal management of cardiovascular disease (CVD) in people with haemophilia (PWH) is a growing issue, given the continuing improvement in life expectancy among PWH. The evolving treatment paradigms targeting higher trough levels and the advent of non-factor replacement therapies (NFRT) means much of the 'protection' PWH were thought to have against CVD may be lost. There is a paucity of evidence regarding the safety of using anticoagulants in PWH. We designed a study assessing the thrombin generation (TG) of PWH of different severities and treatments, compared to non-haemophilia patients receiving a Factor Xa (FXa) inhibitor (apixaban or rivaroxaban), healthy controls, and assessing TG parameters of adding FXa inhibitor to the plasma of PWH receiving emicizumab prophylaxis. In total, 40 patients were included. TG was initiated with 5pM tissue factor (TF) using the calibrated automated thrombinoscope. Compared to those with mild haemophilia, patients receiving a FXa inhibitor had higher endogenous thrombin potential (ETP) (1278.42 vs 1831.36) and velocity index (40.71 vs 112.56), but both had a similar peak height (154.0 vs 262.63) and time to peak (both 5.83). People with severe haemophilia receiving emicizumab had significantly improved TG parameters compared to those not receiving emicizumab - ETP 1678.11 vs 809.96 and peak height 233.8 vs 92.05; however, when FXa inhibitor was added their TG parameters deteriorated to the severe haemophilia range (ETP 1179.60 and peak height 103.05). TG may provide additional useful information regarding the use of anticoagulants in PWH.

INHIBITORS OF INORGANIC POLYPHOSPHATE AND NUCLEIC ACIDS ATTENUATE IN VITRO THROMBIN GENERATION IN PLASMA FROM TRAUMA PATIENTS.

ABSTRACT: Background: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8), and universal heparin reversal agent 8 (UHRA-8). Methods: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 µg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, P < 0.05 significant. Results: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], P < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], P = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], P = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], P < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], P < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all P < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, P < 0.001). Conclusion: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma.

Electrochemical biosensor using direct electron transfer and an antibody-aptamer hybrid sandwich for target detection in complex biological samples.

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer-aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody-antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer-aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody-aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody-aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody-aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer-aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.

Comprehensive investigation of platelet function in patients with cirrhosis.

Cirrhosis presents with thrombocytopenia and possibly thrombocytopathy. Previous studies exploring platelet function gave conflicting results and most controversies are explained by the variety of methods employed for investigation. We sought to assess in-vitro the overall platelet function in cirrhosis. We investigated 34 patients by using the following tests. (i)Aggregometry. (ii)Measurement of the content of platelet granules. (iii)Cytometric platelet activation. (iv)Plasmatic markers of in-vivo platelet activation. (v)Platelet procoagulant activity by thrombin generation (TG) in platelet-rich plasma (PRP). TG measured in PRP for patients and controls was similar. Platelets from patients with cirrhosis showed reduction of aggregation and secretion of ATP. Similar results were observed for platelet activation parameters such as P-selectin expression and PAC-1 platelet binding. Plasma levels of ßeta-thromboglobulin and soluble P-selectin, were increased in patients-vs-controls. In contrast, there were no patients-vs-controls differences for plasmatic platelet-factor-4. Results are consistent with a state of in-vivo platelet activation and decreased in-vitro aggregation. Since bleeding events following invasive procedures are uncommon in cirrhosis, we speculate that in-vitro aggregometry testing does not reflect the situation occurring in-vivo. Results of the study and pathophysiological considerations support the conclusion that platelet function in cirrhosis as determined by aggregometry, although somewhat impaired, may support the overall hemostatic potential, which is needed for most invasive interventions. These conclusions are in line with the recommendations of international guidelines, warning against indiscriminate use of prophylactic preprocedural administration of platelets before invasive procedures. Decision on platelet support should not be made based on in-vitro laboratory testing for platelet function.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection.

Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.

Mathematical models of coagulation-are we there yet?

BACKGROUND: Mathematical models of coagulation have been developed to mirror thrombin generation in plasma, with the aim of investigating how variation in coagulation factor levels regulates hemostasis. However, current models vary in the reactions they capture and the reaction rates used, and their validation is restricted by a lack of large coherent datasets, resulting in questioning of their utility. OBJECTIVES: To address this debate, we systematically assessed current models against a large dataset, using plasma coagulation factor levels from 348 individuals with normal hemostasis to identify the causes of these variations. METHODS: We compared model predictions with measured thrombin generation, quantifying and comparing the ability of each model to predict thrombin generation, the contributions of the individual reactions, and their dependence on reaction rates. RESULTS: We found that no current model predicted the hemostatic response across the whole cohort and all produced thrombin generation curves that did not resemble those obtained experimentally. Our analysis has identified the key reactions that lead to differential model predictions, where experimental uncertainty leads to variability in predictions, and we determined reactions that have a high influence on measured thrombin generation, such as the contribution of factor XI. CONCLUSION: This systematic assessment of models of coagulation, using large dataset inputs, points to ways in which these models can be improved. A model that accurately reflects the effects of the multiple subtle variations in an individual's hemostatic profile could be used for assessing antithrombotics or as a tool for precision medicine.

Are coagulation profiles in Andean highlanders with excessive erythrocytosis favouring hypercoagulability?

Chronic mountain sickness is a maladaptive syndrome that affects individuals living permanently at high altitude and is characterized primarily by excessive erythrocytosis (EE). Recent results concerning the impact of EE in Andean highlanders on clotting and the possible promotion of hypercoagulability, which can lead to thrombosis, were contradictory. We assessed the coagulation profiles of Andeans highlanders with and without excessive erythrocytosis (EE+ and EE-). Blood samples were collected from 30 EE+ and 15 EE- in La Rinconada (Peru, 5100-5300 m a.s.l.), with special attention given to the sampling pre-analytical variables. Rotational thromboelastometry tests were performed at both native and normalized (40%) haematocrit using autologous platelet-poor plasma. Thrombin generation, dosages of clotting factors and inhibitors were measured in plasma samples. Data were compared between groups and with measurements performed at native haematocrit in 10 lowlanders (LL) at sea level. At native haematocrit, in all rotational thromboelastometry assays, EE+ exhibited hypocoagulable profiles (prolonged clotting time and weaker clot strength) compared with EE- and LL (all P < 0.01). At normalized haematocrit, clotting times were normalized in most individuals. Conversely, maximal clot firmness was normalized only in FIBTEM and not in EXTEM/INTEM assays, suggesting abnormal platelet activity. Thrombin generation, levels of plasma clotting factors and inhibitors, and standard coagulation assays were mostly normal in all groups. No highlanders reported a history of venous thromboembolism based on the dedicated survey. Collectively, these results indicate that EE+ do not present a hypercoagulable profile potentially favouring thrombosis.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Impact of therapeutic plasma exchange on intact protein S, apolipoproteins, and thrombin generation.

INTRODUCTION: Therapeutic plasma exchange (TPE), with solvent/detergent (S/D)-treated plasma as replacement fluid, is an extracorporeal blood purification technique with major impact on both coagulation and lipids. Our previous in vitro study showed that S/D-plasma enhances thrombin generation by lowering intact protein S (PS) levels. AIMS: To evaluate the impact of altered lipid balance on coagulation phenotype during heparin-anticoagulated TPE with S/D-plasma, and to investigate whether the lowered intact PS levels with concomitant procoagulant phenotype, are recapitulated in vivo. METHODS: Coagulation biomarkers, thrombin generation with Calibrated Automated Thrombogram (CAT), and lipid levels were measured before and after the consecutive 1st, 3rd and 5th episodes of TPE performed to six patients with Guillain-Barré syndrome or myasthenia gravis. The effects of in vitro dilution of S/D-plasma on thrombin generation were explored with CAT to mimic TPE. RESULTS: Patients did not have coagulation disorders, except elevated FVIII. Intact PS, lipoproteins, especially LDL, Apolipoprotein CIII (ApoC3) and ApoB/ApoA1 ratio declined (p < 0.05). In contrast, VLDL and triglyceride levels stayed intact. CAT lag time shortened (p < 0.05). In vitro dilution of S/D plasma with co-transfused Ringer's lactate and 4% albumin partially reduced its procoagulant phenotype in CAT, which is mainly seen as peak thrombin, and modestly shortened lag time. CONCLUSIONS: After the five settings of TPE using S/D-plasma in vivo, which associated with heparinization and reduced coagulation factor activities, our observations of declining natural anticoagulant intact PS and apolipoproteins refer to rebalance of the hemostatic and lipid profiles.

Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics.

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity.

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 αIIbß3 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 αIIbß3 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.