Clot Contraction Research Articles

Utility of thromboelastography with platelet mapping for monitoring platelet transfusion in qualitative platelet disorders

BackgroundPatients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) exhibit normal platelet counts but impaired platelet aggregation and αIIbβ3 activation. Moderate-to-severe bleeding episodes require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 (Rasgrp2−/− mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Thus, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. ObjectiveWe here studied the usefulness of thromboelastography with platelet mapping (TEG-PM) for ex vivo monitoring of the hemostatic potential in Rasgrp2−/− mice transfused with various amounts of wild-type (WT) platelets. MethodsWhole blood (WB) samples from WT and Rasgrp2−/− mice were tested in TEG-PM and parameters for clot formation and contraction (K time, α-angle, maximum amplitude [MA]) were measured. ResultsRasgrp2−/− WB samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbβ3 activation. Addition of WT platelets improved TEG parameters in a ratio-dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. ConclusionThis proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.

Angiopoietin-like protein 2 inhibits thrombus formation.

Acute myocardial infarction is mainly caused by a lack of blood flood in the coronary artery. Angiopoietin-like protein 2 (ANGPTL2) induces platelet activation and thrombus formation in vitro through binding with immunoglobulin-like receptor B, an immunoglobulin superfamily receptor. However, the mechanism by which it regulates platelet function in vivo remains unclear. In this study, we investigated the role of ANGPTL2 during thrombosis in relationship with ST-segment elevation myocardial infarction (STEMI) with spontaneous recanalization (SR). In a cohort of 276 male and female patients, we measured plasma ANGPTL2 protein levels. Using male Angptl2-knockout and wild-type mice, we examined the inhibitory effect of Angptl2 on thrombosis and platelet activation both in vivo and ex vivo. We found that plasma and platelet ANGPTL2 levels were elevated in patients with STEMI with SR compared to those in non-SR (NSR) patients, and was an independent predictor of SR. Angptl2 deficiency accelerated mesenteric artery thrombosis induced by FeCl3 in Angptl2-/- compared to WT animals, promoted platelet granule secretion and aggregation induced by thrombin and collogen while purified ANGPTL2 protein supplementation reversed collagen-induced platelet aggregation. Angptl2 deficiency also increased platelet spreading on immobilized fibrinogen and clot contraction. In collagen-stimulated Angptl2-/- platelets, Src homology region 2 domain-containing phosphatase (Shp)1-Y564 and Shp2-Y580 phosphorylation were attenuated while Src, Syk, and Phospholipase Cγ2 (PLCγ2) phosphorylation increased. Our results demonstrate that ANGPTL2 negatively regulated thrombus formation by activating ITIM which can suppress ITAM signaling pathway. This new knowledge provides a new perspective for designing future antiplatelet aggregation therapies.

Time-dependent ultrastructural changes during venous thrombogenesis and thrombus resolution

BackgroundDeep vein thrombosis is a common vascular event that can result in debilitating morbidity and even death due to pulmonary embolism. Clinically, patients with faster resolution of a venous thrombus have improved prognosis, but the detailed structural information regarding changes that occur in a resolving thrombus over time is lacking. ObjectivesTo define the spatial-morphologic characteristics of venous thrombus formation, propagation, and resolution at the submicron level over time. MethodsUsing a murine model of stasis-induced deep vein thrombosis along with scanning electron microscopy and immunohistology, we determine the specific structural, compositional, and morphologic characteristics of venous thrombi formed after 4 days and identify the changes that take place during resolution by day 7. Comparison is made with the structure and composition of venous thrombi formed in mice genetically deficient in plasminogen activator inhibitor type 1. ResultsAs venous thrombus resolution progresses, fibrin exists in different structural forms, and there are dynamic cellular changes in the compositions of leukocytes, platelet aggregates, and red blood cells. Intrathrombus microvesicles are present that are not evident by histology, and red blood cells in the form of polyhedrocytes are an indicator of clot contraction. Structural evidence of fibrinolysis is observed early during thrombogenesis and is accelerated by plasminogen activator inhibitor type 1 deficiency. ConclusionThe results reveal unique, detailed ultrastructural and compositional insights along with documentation of the dynamic changes that occur during accelerated resolution that are not evident by standard pathologic procedures and can be applied to inform diagnosis and effectiveness of thrombolytic treatments to improve patient outcomes.

A novel technique to quantify the kinetics of blood clot contraction based on the expulsion of fluorescently labeled albumin into serum

BackgroundThe platelet-driven contraction or retraction of blood clots has been utilized to obtain blood serum for laboratory studies, but now, in vitro clot contraction assays are used in research laboratories and clinics to assess platelet functionality. The static final extent of clot contraction measured using a clot size or expelled serum volume can be supplemented substantially with a dynamic analysis. ObjectivesTo provide a step-by-step protocol for a relatively simple and affordable new automated methodology to follow the kinetics of blood clot contraction, which allows for simultaneous measurements of various samples at a time and requires only a fluorescence plate reader. MethodsThe kinetics of clot contraction in whole blood was assessed by continuously detecting the fluorescence intensity of fluorescein isothiocyanate-albumin added to a blood sample before clotting and expelled into the serum during clot shrinkage. ResultsThe clots are formed and fluorescence is measured in the wells of a black multiwell plate using a standard plate fluorescent reader. The specificity of this technique for clot contraction has been demonstrated by the strong inhibitory effects of blebbistatin, latrunculin A, and abciximab. To validate the new technique, increased fluorescence intensity in the contracting clots was measured in parallel with a visual decrease in clot size performed with the same blood samples. ConclusionThe resulting clot contraction dynamics based on the expulsion of fluorescein isothiocyanate-albumin can be quantified using a number of kinetic parameters as well as a phase kinetics analysis. The advantages and drawbacks of the new technique are discussed.

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction

BackgroundActive and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. ObjectivesTo determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. MethodsUsing the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. ResultsPartial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. ConclusionOur findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.

The effect of nonsteroidal and steroid anti-inflammatory drugs on platelet activation IN VITRO

The article presents the results of a study on the effect of steroid and nonsteroidal anti-inflammatory drugs on platelets.The object of the study was plasma obtained from rat blood (n=10). Blood sampling (2.0 ml from each animal) was carried out from the tail vein into test tubes with sodium citrate. Cell elements were counted directly in blood and in platelet plasma using a MEK-6550 hematological analyzer.The ability to influence platelet activation was determined in drugs such as dexamethasone, ketarol and miloxicam, which were injected into test tubes before plasma reactivation. It was found that the drugs of these groups affect the metabolism of these cells and inhibit their ability to cause clot contraction. It was found that the strength of the negative effect on clot contraction in the steroid drug dexamethasone, which through intermediaries suppresses the activity of phospholipase-A2 and the nonsteroidal drug ketarol, which is the predominant inhibitor of cyclooxygenase of the first type, are similar. It was also found out that the drug meloxicam, which is a predominant inhibitor of cyclooxygenase of the second type, causes only a slight decrease in platelet clot contraction. The results obtained in the experiment indicate a direct positive relationship between the degree of clot contraction and the amount of thromboxane-A2, which is synthesized and released by activated platelets. It is known that the ability of platelets to accelerate and enhance regenerative processes is also directly related to their activation. Based on this, it is suggested that patients who have been prescribed plateletenriched plasma therapy should not use glucocorticoids and preparations with a high degree of selectivity to cyclooxygenase of the first type as anti-inflammatory drugs.

Mechanobiology of Megakaryocyte-Driven Contraction of Plasma Clots

Background: Contractility of non-muscle cells plays a crucial role in various cellular processes including motility, morphogenesis, division, genome replication, intracellular transport, and secretion. Platelet-induced blood clot contraction is an essential process which plays a role in preventing bleeding and in thrombotic disorders. Megakaryocytes (MKs), which are the precursor cells of platelets, share many proteins and structures with platelets. However, little is known about the ability of MKs to contract and interact with extracellular matrix proteins such as fibrin. Previously, the non-muscle myosin II contractility was shown to be essential for MK migration within the bone marrow, to avoid premature proplatelet formation, and to allow branching of proplatelets. Recent studies have identified MKs in lung and brain tissues, as well as associated with thrombi in deceased COVID-19 patients. Notably, patients with severe COVID-19 exhibit elevated MK counts in their blood, indicating potential involvement in hemostasis and thrombosis. Nevertheless, the specific mechanisms by which MKs can contribute to clot contraction remain unknown. Methods: Human induced pluripotent stem cells (iPSC) were differentiated into HPCs followed by expansion to megakaryocytes (iMKs) and analyzed by flowcytometry. Clot formation and contraction were initiated by adding thrombin and calcium to platelet-free-plasma with resuspended iMKs. A combination of real-time macroscale optical tracking, high-resolution confocal microscopy and biomechanical measurements were applied to elucidate the contractile cellular mechanisms and mechanotransduction pathways in iMKs, and ultimately comparing them with platelets. Results: Flowcytometry analysis of unstimulated and thrombin-stimulated iMKs revealed that the major fraction of unstimulated cells expressed αIIbβ3 and GPIbα (~78%), but did not bind PAC-1, a monoclonal antibody specific for the activated conformation of αIIbβ3. Stimulation of iMKs with 1U/ml of thrombin resulted in PAC-1 binding (~68%), indicating activation of aIIbb3. The optical tracking of clot size change over time revealed that thrombin-stimulated iMKs were able to shrink macroscopic plasma clots with biphasic kinetics, similar to platelets. iMK-containing clots and platelet-reach-plasma clots equalized by the total cell surface area demonstrated comparable contraction rates and extent of contraction. The average maximal contractile force per individual iMK in the clot was found to be 145±98nN. In the presence of blebbistatin, the mean final extent and velocity of clot contraction of iMK-containing clots reduced in a dose-dependent manner, in comparison to untreated clots. Pretreatment of iMKs with 1μM latrunculin A, an inhibitor of actin polymerization, completely prevented contraction of plasma clots. In addition, contraction of plasma clots by activated iMKs was abrogated by 10μg/ml abciximab, that prevented binding of fibrin(ogen) to integrin a IIbb 3 receptors. The structural mechanisms of iMK contractility involved formation of filopodia and larger cytoplasmic protrusions undergoing extension-retraction cycles after being attached to surrounding fibrin fibers. Contraction of iMKs caused remodeling of the extracellular fibrin matrix by inducing spatial reorientation of fibrin fibers, accumulation of the fibrin mass on the iMK surface, and compaction of the entire fibrin network. Conclusions: We have demonstrated a hitherto unknown ability of individual MKs to shrink fibrin clots and deciphered the cellular mechanisms of contractility of iPSC-derived MKs. The molecular mechanisms of MK-driven fibrin clot shrinkage were shown to be similar or identical to those of activated platelets, involving non-muscle myosin II activity, actin polymerization, and integrin αIIbβ3-fibrin interactions. Our findings provide a novel mechanistic insight into mechanobiology of MKs that may play a role in modulating the properties of hemostatic blood clots and thrombi. In addition, iMKs can be used as model cells with a potential of genetic modifications to study platelet structure and function, including their contractility.

Qualitative platelet alterations in May — Hegglin anomaly

Introduction: May — Hegglin anomaly is an autosomal dominant mutation in the MYH9 gene that disrupts the dynamics of the cytoskeleton in megakaryocytes. Megakaryocyte dysfunction entails a violation of thrombocytopoiesis, which is manifested by macrothrombocytopenia, sometimes associated with bleeding. Aim: to study the structure and function of platelets in the family members with documented May — Hegglin anomaly. Patients and methods. The proband, her sister and their mother who all had a heterozygous mutation R1933X in the MYH9 gene were examined. The examination included complete blood count, peripheral blood microscopy, platelet flow cytometry, blood clot contraction kinetics, scanning and transmission electron microscopy of platelets. The control group included10 healthy donors. Results. In the proband and her sister the degree of contraction of blood clots was reduced. Unstimulated platelets displayed overexpression of P-selectin and active αIIbβ3 integrin. After TRAP-induced stimulation the proportion of platelets expressing P-selectin in the patients was below normal, indicating partial platelet refractoriness. Electron microscopy of the unstimulated platelets revealed macrothrombocytosis and multiple filopodia, as well as enlargement of the open canalicular system. Conclusion. The May — Hegglin anomaly caused by a heterozygous mutation R1933X in the MYH9 gene is accompanied by thrombocytopenia associated with structural and functional platelet defects. Partial platelet refractoriness and contractile dysfunction can impair the retraction of hemostatic clots, predisposing patients with MYH9-related disorders to bleeding.

Megakaryocyte-induced contraction of plasma clots: cellular mechanisms and structural mechanobiology

AbstractNonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbβ3–fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.

The association between human blood clot analogue computed tomography imaging, composition, contraction, and mechanical characteristics

Background Clot composition, contraction, and mechanical properties are likely determinants of endovascular thrombectomy success. A pre-interventional estimation of these properties is hypothesized to aid in selecting the most suitable treatment for different types of thrombi. Here we determined the association between the aforementioned properties and computed tomography (CT) characteristics using human blood clot analogues. Methods Clot analogues were prepared from the blood of 4 healthy human donors with 5 red blood cell (RBC) volume suspensions: 0%, 20%, 40%, 60% and 80% RBCs. Contraction was measured as the weight of the contracted clots as a percentage of the original suspension. The clots were imaged using CT with and without contrast to quantify clot density and density increase. Unconfined compression was performed to determine the high strain compressive stiffness. The RBC content was analysed using H&E staining. Results The 5 RBC suspensions formed only two groups of clots, fibrin-rich (0% RBCs) and RBC-rich (>90% RBCs), as determined by histology. The density of the fibrin-rich clots was significantly lower (31-38HU) compared to the RBC-rich clots (72-89HU), and the density increase of the fibrin-rich clots was significantly higher (82-127HU) compared to the RBC-rich clots (3-17HU). The compressive stiffness of the fibrin-rich clots was higher (178–1624 kPa) than the stiffness of the RBC-rich clots (6–526 kPa). Additionally, the degree of clot contraction was higher for the fibrin-rich clots (89–96%) compared to the RBC-rich clots (11–77%). Conclusions CT imaging clearly reflects clot RBC content and seems to be related to the clot contraction and stiffness. CT imaging might be a useful tool in predicting the thrombus characteristics. However, future studies should confirm these findings by analysing clots with intermediate RBC and platelet content.

OC 18.3 Platelet Mitochondrial Bioenergetics is Critical for Clot Contraction, Hemostasis and Thrombosis

OC 18.3 Platelet Mitochondrial Bioenergetics is Critical for Clot Contraction, Hemostasis and Thrombosis

PB0879 Structural Mechanisms and Kinetics of Blood Clot Contraction from in Silico Modeling

PB0879 Structural Mechanisms and Kinetics of Blood Clot Contraction from in Silico Modeling

Combined computational modeling and experimental study of the biomechanical mechanisms of platelet-driven contraction of fibrin clots

While blood clot formation has been relatively well studied, little is known about the mechanisms underlying the subsequent structural and mechanical clot remodeling called contraction or retraction. Impairment of the clot contraction process is associated with both life-threatening bleeding and thrombotic conditions, such as ischemic stroke, venous thromboembolism, and others. Recently, blood clot contraction was observed to be hindered in patients with COVID-19. A three-dimensional multiscale computational model is developed and used to quantify biomechanical mechanisms of the kinetics of clot contraction driven by platelet-fibrin pulling interactions. These results provide important biological insights into contraction of platelet filopodia, the mechanically active thin protrusions of the plasma membrane, described previously as performing mostly a sensory function. The biomechanical mechanisms and modeling approach described can potentially apply to studying other systems in which cells are embedded in a filamentous network and exert forces on the extracellular matrix modulated by the substrate stiffness.

Probing interactions of red blood cells and contracting fibrin platelet clots

Contraction of blood clots plays an important role in blood clotting, a natural process that restores hemostasis and regulates thrombosis in the body. Upon injury, a chain of events culminate in the formation of a soft plug of cells and fibrin fibers attaching to wound edges. Platelets become activated and apply contractile forces to shrink the overall clot size, modify clot structure, and mechanically stabilize the clot. Impaired blood clot contraction results in unhealthy volumetric, mechanical, and structural properties of blood clots associated with a range of severe medical conditions for patients with bleeding and thrombotic disorders. Due to the inherent mechanical complexity of blood clots and a confluence of multiple interdependent factors governing clot contraction, the mechanics and dynamics of clot contraction and the interactions with red blood cells (RBCs) remain elusive. Using an experimentally informed, physics-based mesoscale computational model, we probe the dynamic interactions among platelets, fibrin polymers, and RBCs, and examine the properties of contracted blood clots. Our simulations confirm that RBCs strongly affect clot contraction. We find that RBC retention and compaction in thrombi can be solely a result of mechanistic contraction of fibrin mesh due to platelet activity. Retention of RBCs hinders clot contraction and reduces clot contractility. Expulsion of RBCs located closer to clot outer surface results in the development of a dense fibrin shell in thrombus clots commonly observed in experiments. Our simulations identify the essential parameters and interactions that control blood clot contraction process, highlighting its dependence on platelet concentration and the initial clot size. Furthermore, our computational model can serve as a useful tool in clinically relevant studies of hemostasis and thrombosis disorders, and post thrombotic clot lysis, deformation, and breaking.

Citrate Phosphate Dextrose Alters Coagulation Dynamics Ex Vivo

IntroductionCitrate-phosphate-dextrose (CPD) is the most common anticoagulant for blood product storage in the United States. It was developed to prolong shelf life, though there is little research regarding its impact on function following transfusion. We used flow cytometry (FC), thromboelastography (TEG), and a clot contraction assay called the zFlex platform to measure platelet activation and global clot formation in blood samples anticoagulated with either CPD or in a standard blue top citrate (BTC) tube. MethodsSamples were obtained through venipuncture of the antecubital fossa from healthy donors who had not recently taken antiplatelet medication. Samples for FC analysis were spun to obtain platelet-rich plasma, while TEG and zFlex utilized recalcified whole blood. ResultsMean fluorescence intensity for CD62p (P-selectin, marker of platelet activation) in baseline samples was equal, while mean fluorescence intensity in samples activated with thrombin receptor activating peptide was higher in CPD than BTC (65,814 ± 4445 versus 52,483 ± 5435, P = 0.007). TEG results demonstrated similar maximum amplitude for CPD (62.7 ± 1.8 mm versus 61 ± 1 mm) (P = 0.33), though reaction time and kinetics time were significantly longer in CPD versus BTC. CPD R-time: 7.9 ± 0.4 min versus BTC: 3.8 ± 0.4 (P < 0.001). CPD K-time: 2.2 ± 0.2 min versus BTC: 1.6 ± 0.1 min (P < 0.001). Clot contraction strength was not different between the two groups on zFlex: CPD 4353 ± 6 = 517 μN versus BTC 4901 ± 390 μN (P = 0.39). ConclusionsOur findings suggest that CPD does not affect platelet function (minimal difference on FC and no difference in ultimate clot strength, which is ∼80% due to platelet function) but may alter clot dynamics by attenuating thrombin generation.

Platelet glycogenolysis is important for energy production and function

Although the presence of glycogen in platelets was established in the 1960s, its importance to specific functions (i.e., activation, secretion, aggregation, and clot contraction) remains unclear. Patients with glycogen storage disease often present with increased bleeding and glycogen phosphorylase (GP) inhibitors, when used as treatments for diabetes, induce bleeding in preclinical studies suggesting some role for this form of glucose in hemostasis. In the present work, we examined how glycogen mobilization affects platelet function using GP inhibitors (CP316819 and CP91149) and a battery of ex vivo assays. Blocking GP activity increased glycogen levels in resting and thrombin-activated platelets and inhibited platelet secretion and clot contraction, with minimal effects on aggregation. Seahorse energy flux analysis and metabolite supplementation experiments suggested that glycogen is an important metabolic fuel whose role is affected by platelet activation and the availability of external glucose and other metabolic fuels. Our data shed light on the bleeding diathesis in glycogen storage disease patients and offer insights into the potential effects of hyperglycemia on platelets.

Activation of Piezo1 channels in compressed red blood cells augments platelet-driven contraction of blood clots

BackgroundPiezo1 is a mechanosensitive cationic channel that boosts intracellular [Ca2+]i. Compression of red blood cells (RBCs) during platelet-driven contraction of blood clots may cause the activation of Piezo1. ObjectivesTo establish relationships between Piezo1 activity and blood clot contraction. MethodsEffects of a Piezo1 agonist, Yoda1, and antagonist, GsMTx-4, on clot contraction in vitro were studied in human blood containing physiological [Ca2+]. Clot contraction was induced by exogenous thrombin. Activation of Piezo1 was assessed by Ca2+ influx in RBCs and with other functional and morphologic features. ResultsPiezo1 channels in compressed RBCs are activated naturally during blood clot contraction and induce an upsurge in the intracellular [Ca2+]i, followed by phosphatidylserine exposure. Adding the Piezo1 agonist Yoda1 to whole blood increased the extent of clot contraction due to Ca2+-dependent volumetric shrinkage of RBCs and increased platelet contractility due to their hyperactivation by the enhanced generation of endogenous thrombin on activated RBCs. Addition of rivaroxaban, the inhibitor of thrombin formation, or elimination of Ca2+ from the extracellular space abrogated the stimulating effect of Yoda1 on clot contraction. The Piezo1 antagonist, GsMTx-4, caused a decrease in the extent of clot contraction relative to the control both in whole blood and in platelet-rich plasma. Activated Piezo1 in compressed and deformed RBCs amplified the platelet contractility as a positive feedback mechanism during clot contraction. ConclusionThe results obtained demonstrate that the Piezo1 channel expressed on RBCs comprises a mechanochemical modulator of blood clotting that may be considered a potential therapeutic target to correct hemostatic disorders.

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.

Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography

BackgroundThromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported. ObjectivesHere, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples. MethodsClotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer. ResultsWe confirmed the requirement of platelets, platelet contraction, and αIIbβ3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk. ConclusionOur results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.

Physiological correction of hereditary mild hypofibrinogenemia during pregnancy.

Hereditary hypofibrinogenemia is a rare fibrinogen disorder characterised by decreased levels of fibrinogen. Pregnant women with hypofibrinogenemia are at risk of adverse obstetrical outcomes, depending on the fibrinogen level. We investigated how the physiological changes of hemostasis throughout the pregnancy impact the hemostatic balance in a woman with hereditary mild hypofibrinogenemia. Fibrin clot properties were analyzed by turbidimetry and scanning electron microscopy, clot weight and red blood cells retention were measured by whole clot contraction, and in vitro thrombin generation was assessed by calibrated automated thrombogram and ex vivo by TAT. Throughout the pregnancy, the fibrinogen levels increased reaching normal values in the third trimester (activity 3.1g/L, antigen 3.2g/L). In parallel, the fibrin polymerisation increased, the fibrinolysis decreased, the fibrin clot network became denser with thicker fibrin fibers, and the fibrin clot weight and red blood cells retention increased, reaching control's value at the third trimester. Similarly, in vitro and ex vitro thrombin generation increased, reaching maximum values at the delivery. In this case of hereditary mild hypofibrinogenemia we observed a physiological increase of fibrinogen and thrombin generation. Future studies should focus on moderate and severe hypofibrinogenemia, to assess fibrinogen variation and the overall impact of increased TG on the hemostasis balance.