Low Concentrations Of Thrombin Research Articles

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential

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

The Actin Bundling Protein L-Plastin Mediates Platelet Force Generation and Thrombosis

Within minutes of activation, platelets dramatically change their shape, forming filopodia and lamellipodia, aggregate with other platelets or different cells, and can increase their surface area up to 4 times. This relies on a dynamic actin cytoskeleton, regulated by many actin binding proteins in a time- and spatial-specific manner. Deficiency or inhibition of several actin binding proteins in platelets have been shown with impaired platelet spreading and reduced thrombosis. Recently, we found that an actin bundling protein, L-plastin, is expressed in platelets. Its function in platelets has not been studied. In megakaryocytes, we showed that L-plastin is responsible for maintaining cytoskeletal stiffness; in this study we sought to determine L-plastin's role in platelet functions, including thrombosis. We examined the role of L-plastin in platelets in vivo and ex vivo using L-plastin deficient ( Lcp1 -/-) male and female mice. First, we used the collagen/epinephrine induced pulmonary embolism (PE) model and found that survival times were significantly shorter in Lcp1 -/- mice than in their littermate wildtype (WT) controls (Mean±SEM WT 202±12 sec, Lcp1 -/- 139.5±14 sec, P=0.004), suggesting accelerated thrombosis upon L-plastin deficiency in vivo. Second, we examined L-plastin's role in arterial thrombosis in vivo using the laser-induced cremaster arteriole thrombosis mode. Platelets were labeled with anti-GPIb IgG derivative X488 and the thrombosis formation after laser injury was recorded. Preliminary data indicate that thrombi in Lcp1 -/- mice are larger than those in WT mice. Third, we examined L-plastin's role in platelet adhesion using a microfluidics assay with DiOC6-labelled whole blood. At a shear rate of 200/s, the Lcp1 -/- mice had significantly increased platelet adhesion to both collagen and fibrinogen matrices. Fourth, to evaluate the effect of L-plastin on platelet aggregation in vitro, we stimulated washed platelets with 3 different concentrations of thrombin and measured the platelet aggregation (i.e., 0.1, 0.5 and 1 U/mL). When platelets are stimulated with 0.5U/mL or 1U/mL thrombin, platelets from WT and Lcp1 -/- mice showed comparable aggregation kinetics. At 0.1U/mL thrombin, however, preliminary data showed increased aggregation in the Lcp1 -/- group. The difference between the low and high concentration of thrombin indicates potential regulation of the GPIba mediated actin cytoskeleton rearrangement during platelet aggregation. We next examined under low thrombin concentration, the platelet actin cytoskeleton reorganization and platelet spreading. We seeded washed platelets on a fibrinogen matrix with the presence of thrombin (0.1U/mL). Platelets were then fixed and stained for F-actin and imaged using immunofluorescent confocal microscopy. The spreading area per platelet was calculated using FIJI software. Platelets from Lcp1 -/- mice showed significantly increased average membrane area at 30 min (Median WT 689 pixels, Lcp1 -/- 866 pixels, P<0.0001). This suggests that the actin rearrangement in platelets upon fibrinogen activation and thrombin stimulation is inhibited by L-plastin. Lastly, we examined whether L-plastin is involved in platelet force generation during the actin reorganization. We used a recently developed “black dot” assay. Briefly, a fluorescent pattern (i.e., black dots) was microcontact printed onto a flexible polydimethylsiloxane substrate and coated with fibrinogen. Platelets were seeded onto the matrix and fixed after 30 min. The contractile forces of individual platelets were measured based on the deformation of the fluorescent micropattern as illustrated in Fig 1A. The contractile force in platelets from Lcp1 -/- mice is decreased with altered actin distribution pattern compared to WT mice (Fig 1. Contractile force per platelet, Mean±SEM WT 14.78±0.86nN, Lcp1 -/- 12.03±0.67nN, P=0.01). In summary, we found accelerated thrombosis in Lcp1 -/- mice, associated with accelerated platelet adhesion and spreading ex vivo. Mechanistically, our preliminary data suggest this is due to impaired actin bundle formation and decreased platelet contractile force. L-plastin may be unique among actin binding proteins.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Inhibitory Effects of Aspirin and Cilostazol on Intracellular Ca<sup>2+</sup> Mobilization and Aggregation in Thrombin-activated Human Platelets

Platelets play an important role in physiological hemostatic mechanisms. In contrast, platelet activation has been implicated in pathological conditions, such as atherosclerosis, angiogenesis, and inflammation. Thrombin is considered to be of particular pathological importance as a platelet-activating substance, and thrombin-activated platelets are detected in the blood of patients with advanced occlusive arterial disease. Ca2+ acts as a second messenger in platelet activation, and the regulation of intracellular Ca2+ concentrations ([Ca2+]i) is important for controlling platelet functions. However, changes in [Ca2+]i by antiplatelet agents remain unclear. Therefore, we herein investigated the relationship between [Ca2+]i and the intensity of platelet aggregation after a thrombin stimulation, the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents, and the effects of antiplatelet agents on thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. Fura2-loaded platelets were treated with phosphate-buffered saline or a low concentration of thrombin (0.005 U/mL), followed by antiplatelet agents (aspirin or cilostazol), and changes in [Ca2+]i and the intensity of platelet aggregation by the thrombin stimulation were measured using fluorescence spectrophotometry. Changes in [Ca2+]i and the intensity of platelet aggregation after the thrombin stimulation as well as the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents indicated that cilostazol exerted stronger antiplatelet effects than aspirin and also that antiplatelet effects may be attenuated in thrombin-activated platelets. The present results also suggest the utility of thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. These results may contribute to future drug development for antiplatelet therapy. J. Med. Invest. 70 : 94-100, February, 2023.

ADP receptor P2Y12 is the capstone of the cross-talk between Ca2+ mobilization pathways dependent on Ca2+ ATPases sarcoplasmic/endoplasmic reticulum type 3 and type 2b in platelets

BackgroundBlood platelet Ca2+ stores are regulated by 2 Ca2+-ATPases (SERCA2b and SERCA3). On thrombin stimulation, nicotinic acid adenosine dinucleotide phosphate mobilizes SERCA3-dependent stores, inducing early adenosine 5‘-diphosphate (ADP) secretion, potentiating later SERCA2b-dependent secretion. ObjectivesThe aim of this study was to identify which ADP P2 purinergic receptor (P2Y1 and/or P2Y12) is(are) involved in the amplification of platelet secretion dependent on the SERCA3-dependent Ca2+ mobilization pathway (SERCA3 stores mobilization) as triggered by low concentration of thrombin. MethodsThe study used the pharmacologic antagonists MRS2719 and AR-C69931MX, of the P2Y1 and P2Y12, respectively, as well as Serca3-/- mice and mice exhibiting platelet lineage-specific inactivation of the P2Y1 or P2Y12 genes. ResultsWe found that in mouse platelets, pharmacological blockade or gene inactivation of P2Y12 but not of P2Y1 led to a marked inhibition of ADP secretion after platelet stimulation with low concentration of thrombin. Likewise, in human platelets, pharmacological inhibition of P2Y12 but not of P2Y1 alters amplification of thrombin-elicited secretion through SERCA2b stores mobilization. Finally, we show that early SERCA3 stores secretion of ADP is a dense granule secretion, based on parallel adenosine triphosphate and serotonin early secretion. Furthermore, early secretion involves a single granule, based on the amount of adenosine triphosphate released. ConclusionAltogether, these results show that at low concentrations of thrombin, SERCA3- and SERCA2b-dependent Ca2+ mobilization pathways cross-talk via ADP and activation of the P2Y12, and not the P2Y1 ADP receptor. The relevance in hemostasis of the coupling of the SERCA3 and the SERCA2b pathways is reviewed.

Differential Inhibition of Platelet Reactivity by Dual Therapy With Aspirin and Low-Dose Rivaroxaban in Peripheral Arterial Disease: A Pilot Study.

Patients with peripheral arterial disease (PAD) benefit from combination therapy with acetylsalicylic acid (ASA, 100 mg, one time per day) plus low-dose rivaroxaban (2.5 mg, two times per day) compared to ASA monotherapy. In particular, major adverse cardiac and limb events were significantly reduced after peripheral endovascular revascularization (EVR). In this pilot study, the platelet activation status in vivo and platelet reactivity in vitro were longitudinally analyzed by flow cytometric assays and calibrated automated thrombography in platelet-rich plasma (PRP) from 10 patients with PAD receiving ASA (100 mg, one time per day) before EVR, ASA plus clopidogrel (75 mg, one time per day) after EVR, and ASA plus rivaroxaban (2.5 mg, two times per day) during a long-term follow-up. Platelet responsiveness to clopidogrel was compared to additional 10 patients with stable PAD and clopidogrel (75 mg, one time per day) monotherapy. ASA plus rivaroxaban treatment resulted in a significantly decreased thrombin peak in PRP for two triggers, namely, low concentration of tissue factor (TF) and thrombin, compared to ASA monotherapy. TF-controlled thrombin generation was additionally characterized by a significantly prolonged lag time in PRP and platelet-free plasma during ASA plus rivaroxaban combination therapy. In comparison, ASA plus clopidogrel treatment presented a significant reduction of the thrombin peak in PRP, which was less pronounced than during subsequent ASA plus rivaroxaban therapy. Platelet responsiveness to clopidogrel was observed for 60% of patients receiving ASA plus clopidogrel and clopidogrel monotherapy, respectively. Blocking of CD36 on the platelet surface further reduced the thrombin peak in PRP induced by TF for all three therapy regimes. Platelet activation in vivo and in response to the GPVI-agonist convulxin or thrombin in vitro was similar, whereas integrin αIIbβ3 activation and α-granule release induced by the PAR-1 activating peptide TRAP-6 were significantly diminished during ASA plus rivaroxaban treatment compared to ASA monotherapy. In conclusion, the data of this pilot study indicate an inhibitory effect of rivaroxaban on the thrombin propagation phase of CD36-sensitive platelet thrombin formation in patients with PAD treated with ASA plus rivaroxaban combination therapy, which is associated with decreased PAR-1 but not thrombin-mediated platelet activation.

Novel Au-Se Nanoprobes for Specific Thrombin Detection in Diagnosis of Lung Cancer.

Thrombin is associated with malignant tumors and promotes tumor development, metastasis, and angiogenesis, therefore its identification especially in lung cancer cells is crucial. Because the interference of in vivo biothiols caused false positive findings with prior gold fluorescent nanoprobes, in this manuscript, an Au-selenol(Se) nanoprobe (5-FAM-peptide-Se-AuNPs) that could specifically detect thrombin was designed and compared to traditional Au-S nanoprobes. For reaching this goal, fluorophore-bearing thrombin-specific peptide containing selenol at the end was synthesized. The nanoprobe may be broken by thrombin to regain its fluorescence in lung cancer cells, allowing for high-sensitivity thrombin detection. Since the Au-Se bond is more stable than the Au-S bond, the accuracy of the detection results can be guaranteed. The probe synthesis method is simple and cost-effective, as well as having high biocompatibility. Low concentrations of thrombin can be detected and imaged in lung cancer cells. The synthetic method of this probe opens up new avenues for the application of Au-Se bonds.

Effect of Low Concentrations of Thrombin on the Dynamic Surface Properties of Fibrinogen Solutions

Effect of Low Concentrations of Thrombin on the Dynamic Surface Properties of Fibrinogen Solutions

Q94 is not a selective modulator of proteinase-activated receptor 1 (PAR1) in platelets

Thrombin is a potent platelet activator, acting through proteinase-activated receptors −1 and −4 (PAR1 and PAR4). Of these, PAR-1 is activated more rapidly and by lower thrombin concentrations. Consequently, PAR-1 has been extensively investigated as a target for anti-platelet drugs to prevent myocardial infarction. Q94 has been reported to act as an allosteric modulator of PAR1, potently and selectively inhibiting PAR1-Gαq coupling in multiple cell lines, but its effects on human platelet activation have not been previously studied. Platelet Ca2+ signaling, integrin αIIbβ3 activation and α-granule secretion were monitored following stimulation by a PAR1-activating peptide (PAR1-AP). Although Q94 inhibited these responses, its potency was low compared to other PAR1 antagonists. In addition, αIIbβ3 activation and α-granule secretion in response to other platelet activators were also inhibited with similar potency. Finally, in endothelial cells, Q94 did not inhibit PAR1-dependent Ca2+ signaling. Our data suggest that Q94 may have PAR1-independent off-target effects in platelets, precluding its use as a selective PAR1 allosteric modulator.

Design of a protein-targeted DNA aptamer using atomistic simulation

The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such approach involves the formation of osmium tetroxide 2,2'-bipyridine protein adducts, followed by electrochemical detection of analytes that bind specifically to electrode-tethered aptamers. In conjunction with a 27-mer DNA aptamer that binds specifically to exosite II on human alpha thrombin, this technique permits, in theory, a highly sensitive diagnostic tool for the quantification of serum thrombin levels. However, thrombin's aptamer binding site is lined by two tryptophan residues and the conjugation of bulky osmium groups to these residues weakens aptamer binding by an estimated 4 to 12 kcal/mol, undermining detection sensitivity. Therefore, we have rationally modified this DNA aptamer to strengthen its thrombin binding in the presence of conjugated osmium. Specifically, aptamers carrying long hydrophobic thymine derivatives in place of guanine 21 have binding affinities for osmium-conjugated thrombin that are enhanced by 10 to 15 kcal/mol, suggesting that these modified aptamers may be effective in a highly sensitive electrochemical sensor for the quantification of low concentrations of thrombin. Our approach of using molecular simulation to subtly re-engineer a DNA aptamer may be generally applicable for the optimization of other macromolecular binding interfaces. Communicated by Ramaswamy H. Sarma

Protection of β2GPI Deficient Mice from Thrombosis May Reflect a Platelet Activation Defect

IntroductionAntiphospholipid syndrome (APS) is an autoimmune disorder caused by “antiphospholipid” antibodies (aPL) directed against β2-glycoprotein I (β2GPI). Although β2GPI is proposed to have both anti- and procoagulant properties in vitro, its physiological role in coagulation in vivo is not well understood. Previous studies have shown that β2GPI deficient mice display impaired thrombin generation but failed to demonstrate an anticoagulant role in vivo. Recent findings from our laboratory demonstrated that β2GPI-deficient mice (APOH -/-) developed by CRISPR/Cas9 had shown delayed time to thrombosis as evidenced by prolonged clotting times in carotid artery occlusion models. However, no mechanism was identified for the delayed time to thrombosis phenotype.MethodsVenous thrombosis by complete occlusion of the IVC was performed as described previously (Wrobleski et al., 2011). In brief, an abdominal incision was made on anesthetized mice to visualize IVC. The activated partial thromboplastin time (aPTT); prothrombin time (PT) and Tissue factor-induced thrombin generation time (TGT) were performed as described previously (Stavrou et al., 2014). Protein C activity was performed according to manufacturer's recommendations (Chromogenix Coamatic® Protein C, Diapharma). To assess platelet activation, platelets were isolated under resting conditions using retro-orbital blood and the final washed platelet pellet was resuspended in Tyrodes solution. Platelets were stimulated with 0.25 U/ml thrombin and incubated with 1 µL of CD62P-FITC or Oregon Green conjugated-fibrinogen for 30 minutes. Platelets were fixed with 100 μL 2% formalin, and quantification of platelet surface P-selectin and fibrinogen binding to activated platelet GPIIb/IIIa was performed by flow cytometry (Accuri Flow Cytometer, BD Biosciences). For mouse tail vein bleeding time assays, mice were anesthetized and the tail transected approximately 5 mm from the tip. The tail was then placed in a 50 ml falcon tube containing saline pre-warmed to 37°C. The time to cessation of bleeding was determined visually. All experiments were performed on 8-12 week old mice.ResultsaPTT and PT results for APOH +/+ and APOH -/- mice were 41.82 ± 1.174 and 41.38 ± 2.026 seconds, and 14.55 ± 2.262 and 11.30 ± 0.578 seconds, respectively (NS). After IVC ligation, thrombi in APOH -/- mice had a mean weight of 16.94 ± 1.782 mg compared to 27.69 ± 1.725 mg in APOH +/+ mice littermates (P = 0.0002, Figure A). Thrombin generation induced by either tissue factor or aPPT reagent showed peak thrombin generation at 15 min with no difference between APOH +/+ and APOH -/- mice. Likewise, no significant differences were observed in percent Protein C activity levels between APOH +/+ (8.058 ± 1.433) and APOH -/- (6.453 ± 0.924).Stimulation with 0.25 U/ml thrombin resulted in significantly greater expression of P-selectin) on platelets of APOH +/+ (161.3 ± 30.62 MFI) compared to those from APOH -/- mice (54.47 ± 9.721 MFI) (P= 0.0101;Figure 1B). Likewise, there was significantly greater fibrinogen binding to stimulated platelets from APOH +/+ (129.7 ± 32.21 MFI) compared to APOH-/- mice (35.30 ± 2.144 MFI) with P = 0.0111 (Figure 1C). Finally, consistent with platelet activation studies, tail vein bleeding times were mildly, but significantly prolonged in APOH -/- mice (192.3 ± 45.86 sec) compared to APOH+/+ mice (95.14 ± 9.582 sec) with P = 0..ConclusionThe effects of β2GPI, if any, on coagulation processes is controversial, and has not been thoroughly studied in β2GPI deficient animals. The results presented here suggest that the smaller thrombi that form in mice deficient in β2GPI following IVC occlusion may reflect a mild platelet function defect. This hypothesis is supported by diminished P-selectin expression and fibrinogen binding in response to 0.25 U/ml thrombin by platelets from β2GPI deficient mice compared to wild-type littermates. Moreover, tail vein bleeding times were mildly prolonged in β2GPI deficient mice. Taken together, these studies suggest that β2GPI may actually contribute to hemostasis by supporting platelet responses to low concentrations of thrombin. Additional studies are needed to characterize these effects in detail. [Display omitted] DisclosuresMcCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

The Reevaluation of Thrombin Time Using a Clot Waveform Analysis.

Object: Although thrombin burst has attracted attention as a physiological coagulation mechanism, clinical evidence from a routine assay for it is scarce. This mechanism was therefore evaluated by a clot waveform analysis (CWA) to assess the thrombin time (TT). Material and Methods: The TT with a low concentration of thrombin was evaluated using a CWA. We evaluated the CWA-TT of plasma deficient in various clotting factors, calibration plasma, platelet-poor plasma (PPP), and platelet-rich plasma (PRP) obtained from healthy volunteers, patients with thrombocytopenia, and patients with malignant disease. Results: Although the TT-CWA of calibration plasma was able to be evaluated with 0.01 IU/mL of thrombin, that of FVIII-deficient plasma could not be evaluated. The peak time of CWA-TT was significantly longer, and the peak height significantly lower, in various deficient plasma, especially in FVIII-deficient plasma compared to calibration plasma. The second peak of the first derivative (1st DP-2) was detected in PPP from healthy volunteers, and was shorter and higher in PRP than in PPP. The 1st DP-2 was not detected in PPP from patients with thrombocytopenia, and the 1st DP-2 in PRP was significantly lower in patients with thrombocytopenia and significantly higher in patients with malignant disease than in healthy volunteers. Conclusion: The CWA-TT became abnormal in plasma deficient in various clotting factors, and was significantly affected by platelets, suggesting that the CWA-TT may be a useful test for hemostatic abnormalities.

Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Abstract Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Expression of Functional Human Proteinase Activated Receptor (PAR)-1 on Mouse Platelets

Thrombin activates platelets by specific cleavage of PARs leading to platelet aggregation. Human (h) platelets express PAR-1 and PAR-4, each with a different sensitivity to thrombin and specific kinetics of activation/deactivation influencing thrombogenesis and thrombus stability. PAR-1 and PAR-4 are candidates for the generation of anti-thrombotic drugs; indeed, a PAR-1 inhibitor (Vorapaxar) is already in clinical use. However, side effects such as bleeding warrant a continuous effort towards the generation of novel alternative inhibitors. In this regard, human PAR-4 may be a more selective antithrombotic target with a lesser impact on hemostasis. Mice are commonly used to test anti-thrombotic drugs and their mechanisms of action, but cannot be used in this instance because mouse (m) platelets express different PAR subtypes than human, i.e. PAR-3 and PAR-4. Moreover, mPAR-3 cannot be cleaved by thrombin and only acts as a co-receptor enhancing mPAR-4 response. In human platelets, PAR-1 and PAR-4 can act independently of one another. Yet, a different platelet membrane receptor, unrelated to PARs, which binds thrombin with high affinity - glycoprotein (GP) Ibα in the GPIb-IX-V complex - enhances PAR-1 response to low-dose thrombin through a mechanism only partially elucidated. Humanizing mouse platelets with respect to PAR expression would greatly facilitate explaining the functional interplay of GPIb with PAR-1 and PAR-4 and, notably, the role of these different PARs in hemostasis and thrombosis. Attempts to introduce hPAR-1 in mouse platelets have been so far unsuccessful (French et al. 2016, Arachiche et al. 2014) and claims have been made that obstacles to achieve this goal may be unsurpassable. Using a different approach, we introduced a floxed hPAR-1 transgene in the ROSA26 locus of C57BL6/J mice under the strong CAG promoter. The presence of an IRES sequence and a second transgene for eGFP allowed us to screen efficiently for platelets expressing hPAR1. Mice positive for the floxed-transgene were then bred with either a germ-line Cre recombinase-expressing strain (EIIa-Cre) or one in which Cre expression was linked to a platelet specific receptor (PF4-Cre). The percentage of platelets isolated from EIIa-Cre/PAR1+/- mice expressing eGFP was between 35 to 40% and breeding to homozygosity did not increase transgene expression. In contrast, eGFP expression was positive in nearly all PF4-Cre/PAR1+/-mice, suggesting that suppression of hPAR1 expression at the germ-line level could be advantageous for megakaryocyte development. As a functional test for transgene-expressed hPAR-1 we measured aggregation and intracellular calcium increase in washed platelets from PF4-Cre/PAR1+/+ mice stimulated by 5 μmol/liter of a specific PAR-1 activation peptide (TFLLRN, P1-AP). In both assays, the response of human or hPAR-1 expressing mouse platelets was indistinguishable; littermate control platelets lacking transgene expression (floxed hPAR1) did not respond to a 10 times higher concentration of peptide. Interestingly, when stimulated with a low dose of thrombin (0.25 nM) aggregation and intracellular calcium increase of PF4-Cre/PAR1+/+ platelets was similar to littermate controls. We reasoned that this could be a consequence of the presence of mPAR3, owning to its high affinity for thrombin. Thus we proceeded to cross PF4-Cre/PAR1+/+ with mPAR3-/- mice, so to obtain mice that express only mPAR4 and hPAR1. Aggregation induced with P1-AP (5 μM) was similar with platelets expressing PF4-Cre/PAR1+/+ and mPAR3-/-/PF4-Cre/PAR1+/+. As expected from the cofactor role of mPAR3, the minimal amount of thrombin necessary to aggregate fully platelets from mPAR3-/-/PF4-Cre/PAR1-/- was higher than in wild-type mice (2 nM vs 0.25 nM); however, 2 nM was also the minimum thrombin concentration necessary to activate mPAR3-/-/PF4-Cre/PAR1+/+ platelets. Therefore, the presence of a functional hPAR1 responsive to P1-AP does not influence the response of mouse platelets to low thrombin concentrations mediated by mPAR4, indicating that hPAR-1 and mPAR-4, like the human homologue, respond to thrombin independently of one another. In conclusion, to date we have shown that expression of a functional hPAR1 in mouse platelets is possible and should provide a versatile animal model to address several open questions on the role of platelet PAR-1 and GPIbα in hemostasis and thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Acetyl-CoA carboxylase inhibition alters tubulin acetylation and aggregation in thrombin-stimulated platelets

Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14 C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production, which could result from a decreased NOX2 activity. Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway.

Role of Thrombin in Central Nervous System Injury and Disease.

Thrombin is a Na+-activated allosteric serine protease of the chymotrypsin family involved in coagulation, inflammation, cell protection, and apoptosis. Increasingly, the role of thrombin in the brain has been explored. Low concentrations of thrombin are neuroprotective, while high concentrations exert pathological effects. However, greater attention regarding the involvement of thrombin in normal and pathological processes in the central nervous system is warranted. In this review, we explore the mechanisms of thrombin action, localization, and functions in the central nervous system and describe the involvement of thrombin in stroke and intracerebral hemorrhage, neurodegenerative diseases, epilepsy, traumatic brain injury, and primary central nervous system tumors. We aim to comprehensively characterize the role of thrombin in neurological disease and injury.

Pitfalls in the assessment of disseminated intravascular coagulation in patients on dabigatran

Dabigatran is an orally administrated anticoagulant that directly inhibits thrombin. However, the drug can affect routine coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT), as well as haemostasis assays, (e.g., clot-based coagulation factor assays). There are limited data on the effect of dabigatran on some fibrinogen measurements and on D-dimer assays, both important components in the laboratory assessment of disseminated intravascular coagulation (DIC). The objectives of this study were: (1) to determine the effects of various concentrations of dabigatran on fibrinogen and D-Dimer assays; and (2) to compare the von Clauss method of fibrinogen measurement using two reagents with differing thrombin concentrations (35 UNIH/mL and 100 UNIH/mL) and PT-derived fibrinogen measurement in the presence of the drug. Aliquots of pooled normal plasma were spiked with different concentrations of dabigatran to reflect invivo on-therapy levels as well as levels observed in cases of massive accumulation of the drug. Of the routine coagulation assays, in ascending order of sensitivity to dabigatran were PT, APTT and TT. The von Clauss method of measuring fibrinogen using a reagent with low thrombin concentration was affected even at drug levels corresponding to invivo trough concentrations, whereas the reagent with higher thrombin concentration was only affected at drug levels that were above observed peak concentrations in patients taking 150 mg of the drug twice daily. PT-derived fibrinogen was affected at approximately invivo peak drug concentrations. The D-dimer assay was affected only at drug concentrations well above peak drug levels. Attempts at invitro neutralisation of the drug with DOAC-Stop resulted in 'correction' of some of these measurements depending on drug concentration. Like the routine coagulation assays, there is a dabigatran concentration dependent effect on the accuracy of fibrinogen and D-dimer assays. Falsely low fibrinogen results due to dabigatran may confound the assessment of DIC and diagnostic laboratories need to evaluate the performance of their own reagents.

Thrombin-Loaded TA-CaCO3 Microspheres as a Budget, Adaptable, and Highly Efficient Hemostatic

Fast hemostasis, biosecurity, and adaptation of different bleeding sites are the major challenges for hemostatic materials. In this work, an easy-to-make, rapid hemostatic, tannic acid doped calcium carbonate microsphere with a low concentration of thrombin (Thr@TA-CaCO3) was developed. In an in vitro blood clotting assay, the hemostatic effect of Thr@TA-CaCO3 varied with the change of thrombin amount. Furthermore, the average hemostatic time can be reduced from 205 to 20 s as the thrombin levels are elevated. When applied in different hemorrhage models, the Thr@TA-CaCO3-treated group had a lower blood loss than the tannic acid modified CaCO3 (TA-CaCO3) groups. Especially in the rabbit artery injury model, the total bleeding amount in the Thr@TA-CaCO3-treated group was only 11.97% of that in the TA-CaCO3 groups, proving that Thr@TA-CaCO3 had an excellent hemostatic effect even in the case of rapidly flowing blood. Based on the above, Thr@TA-CaCO3 is a promising hemostatic agent and can be applied in more extensive clinical applications.

Loss of Tet2 affects platelet function but not coagulation in mice.

Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies. TET2 mutations were found in a variety of hematological disorders such as CMML (60%), MDS (30%), MPN (13%) and AML (20%). Interestingly, it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation. However, the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined. Here in this study, using a genetically engineered Tet2 deletion mouse model, we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes. Additionally, Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin, although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function. Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.

The Role of Thrombin in Brain Injury After Hemorrhagic and Ischemic Stroke.

Thrombin is increased in the brain after hemorrhagic and ischemic stroke primarily due to the prothrombin entry from blood either with a hemorrhage or following blood-brain barrier disruption. Increasing evidence indicates that thrombin and its receptors (protease-activated receptors (PARs)) play a major role in brain pathology following ischemic and hemorrhagic stroke (including intracerebral, intraventricular, and subarachnoid hemorrhage). Thrombin and PARs affect brain injury via multiple mechanisms that can be detrimental or protective. The cleavage of prothrombin into thrombin is the key step of hemostasis and thrombosis which takes place in every stroke and subsequent brain injury. The extravascular effects and direct cellular interactions of thrombin are mediated by PARs (PAR-1, PAR-3, and PAR-4) and their downstream signaling in multiple brain cell types. Such effects include inducing blood-brain-barrier disruption, brain edema, neuroinflammation, and neuronal death, although low thrombin concentrations can promote cell survival. Also, thrombin directly links the coagulation system to the immune system by activating interleukin-1α. Such effects of thrombin can result in both short-term brain injury and long-term functional deficits, making extravascular thrombin an understudied therapeutic target for stroke. This review examines the role of thrombin and PARs in brain injury following hemorrhagic and ischemic stroke and the potential treatment strategies which are complicated by their role in both hemostasis and brain.