Clot Contraction Research Articles

Platelets loaded with liposome‐encapsulated thrombin have increased coagulability

Background Platelets are integral to clot formation and are often transfused to stop or prevent bleeding. However, transfusions of platelets are not always effective, particularly in the most severe cases of hemorrhage. Nanoparticle systems have been developed to mimic platelets but inherently lack important aspects of platelet function, which limits their potential effectiveness. Objectives Increasing the natural coagulability of transfusable platelets could increase their efficacy during treatment of severe hemorrhage. Thrombin is a potent platelet agonist that currently cannot be used intravenously because of the risk of thrombosis. We hypothesized that delivery of thrombin to ex vivo platelets via liposomal encapsulation would enable transfusable platelets to become more coagulable in response to platelet agonists. Methods Thrombin was encapsulated into nanoliposomes and delivered to platelets ex vivo. Platelet coagulability was measured by monitoring platelet activation, clot contraction, clot time and clot stability in several in vitro assays. These parameters were also measured under conditions where coagulation is compromised, including during acidosis, antiplatelet drugs, hemophilia A and trauma-induced coagulopathy. Results Liposomal thrombin was endocytosed and used by platelets ex vivo but was not secreted upon activation. These modified platelets became more sensitive and responsive to agonists and improved clotting time even under conditions that normally cause platelet dysfunction or have impaired coagulation. Conclusions Several aspects of platelet function were enhanced by ex vivo delivery of liposomal thrombin.

Abstract 525: Contraction of Blood Clots Influences Their Susceptibility to Fibrinolysis

The success of endogenous and therapeutic fibrinolysis is governed in part by the structure of the fibrin clot. An understudied feature that potentially affects fibrinolysis is the degree of platelet-driven shrinkage, i.e. contraction, of blood clots and thrombi. The aim of this work was to examine the effect of clot contraction on the rate of naturally occurring fibrinolysis from within and external fibrinolysis to simulate therapeutic thrombolysis. Clot contraction, which was initatied with 2mM CaCl 2 and 1U/ml thrombin, was impaired by ~75% by inhibiting platelet non-muscle myosin IIa activity (blebbistatin), actin polymerization (latrunculin A), and platelet-fibin(ogen) binding (abciximab). Internal fibrinolysis measured using dynamic optical tracking of clot size as a function of time occurred 2X faster in contracted clots compared to uncontracted clots in the presence of 75ng/ml tPA. In direct contrast, the dynamic release of radioactive fibrin degradation products as a measure of external fibrinolysis was 4X faster in uncontracted clots when 75 ng/ml tPA was added to the surface of preformed clots. This difference in the susceptibility of contracted and uncontracted clots to internal versus external lysis suggests that the lysis rate is dominated by the interplay of clot permeability to fibrinolytic enzymes as well as the spatial proximity of the fibrin fibers themselves. These results have implications for understanding clot stability in patients with thrombotic disorders and improving clot lysis by impairing platelet-mediated clot contraction.

Abstract 379: Platelet-driven Contraction of Venous Thrombi Modulates Their Obstructiveness and Embologenicity

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in clots made from the blood of 55 patients with VTE not receiving antiplatelet and anticoagulant medications. In addition, we studied the ultrastructure of ex vivo venous thrombi, as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo . The extent and rate of contraction of in vitro clots were reduced by 2-fold in clots from the blood of VTE patients compared to healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared to that of those with isolated venous thrombosis, suggesting that less compacted thrombi may be prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness to stimulation. Morphologically, 75% of platelets from VTE patients were spontaneously partially activated (with filopodia) compared to only 21% of those from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared to platelets from healthy individuals. The results obtained suggest that the impaired contraction of thrombi resulting from platelet dysfunction is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi. Furthermore, an assay for clot contraction may have diagnostic and prognostic value for venous thromboembolism. The work was supported by the Program for Competitive Growth at Kazan Federal University.

Morphometric analysis of spread platelets identifies integrin \u03b1IIb\u03b23-specific contractile phenotype

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin αIIbβ3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner. The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of αIIbβ3 integrins. Cytoskeletal morphologies in vitro thus inform about platelet adhesion receptor identity and functionality, and integrin αIIbβ3 mechanotransduction fundamentally determines the adoption of a bipolar phenotype associated with contraction. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet behaviour contributing to elusive bleeding phenotypes.

Erythrocyte compression index is impaired in patients with residual vein obstruction

Defective clot contraction has been postulated to contribute to thrombosis. We aimed to evaluate the association of residual vein obstruction (RVO) with erythrocyte compression within the whole-blood clot. We studied 32 patients with venous thromboembolism (VTE) taking vitamin K antagonists (VKAs) for at least 3 months (median time in therapeutic range 60%), including 12 (37.5%) with RVO, and 32 age- and sex-matched controls. In all study participants we evaluated whole blood clot retraction, expressed as the erythrocyte compression index (ECI), defined as a ratio of mean polyhedrocyte area to mean native erythrocyte area, along with clot area covered by polyhedrocytes, plasma clot permeability (Ks), clot lysis time (CLT), and thrombin generation. In both groups higher ECI, indicating impaired clot contraction, increased with older age, higher body mass index, red blood cell distribution width, and lower platelet count (all p < 0.05), but not with red blood cell count. In VTE patients ECI was 15.8% higher than in controls (median 63.6 vs. 54.9%, p = 0.021). Subjects with RVO had 20% higher ECI and 155% lower clot area covered by polyhedrocytes. RVO patients had also prolonged CLT by 41%, but not Ks, and elevated peak thrombin generation by 33%, as compared to those without RVO (all p < 0.05). This study is the first to show impaired compression of erythrocytes in RVO patients despite VKA anticoagulation. Altered ECI coexisted with hypolysability and increased thrombin generation. ECI might be useful in the diagnostic process of RVO or post-thrombotic syndrome and can help optimize the anticoagulant therapy.

A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.

Structural and Biomechanical Changes During Platelet-Driven Clot Contraction

Blood clot contraction plays an important role in prevention of bleeding and in thrombotic disorders. We found that activated platelets bend and shorten individual fibrin fibers via their filopodia that undergo sequential extension and retraction, as if pulling hand-over-hand. Platelets also induce compaction of fibrin fibers into platelet-attached agglomerates. As a result of simultaneous pulling on multiple, closely-set fibrin fibers, platelets pull themselves closer to each other and form secondary clusters larger than the initial aggregates. Contracting platelets actively remodel a fibrin network by increasing its density followed by enhanced clot stiffness. Kinetic analysis of the time course of structural and mechanical transitions revealed a multiphasic behavior with at least three distinct phases that differ in duration and rate constants. All the observed changes were reduced or abrogated in the presence of specific inhibitors of non-muscle myosin IIA (blebbistatin) and the platelet integrin αIIbβIII (abciximab), indicating that actomyosin-driven cell contractility and integrin-fibrin mediated platelet-fibrin interactions are crucial for contraction of blood clots. Finally, blood clot contraction was found to be a spatially non-uniform process with faster compression of the clot edge and a delayed deformation of the clot interior. Altogether, the results provide a quantitative structural basis for the mechanobiology of clot contraction at various spatial scales from a single cell/single fiber level up to the network and macroscopic levels. Our results obtained on platelet-induced contraction of the filamentous fibrin network are of fundamental importance because they provide a foundation for understanding dynamic and complex biomechanical interplay between non-muscle cells and fibrous extracellular matrices of various compositions.

Blood Clot Contraction is Reduced in Sickle Cell Disease due to Increased Rigidity of Erythrocytes

Blood Clot Contraction is Reduced in Sickle Cell Disease due to Increased Rigidity of Erythrocytes

Impaired contraction of blood clots as a novel prothrombotic mechanism in systemic lupus erythematosus.

The aim of this work was to examine a possible role of clot contraction/retraction in thrombotic complications of systemic lupus erythematosus (SLE). Using a novel automated method, we investigated kinetics of clot contraction in the blood of 51 SLE patients and 60 healthy donors. The functionality of platelets in the SLE patients was assessed using flow cytometry by expression of P-selectin and fibrinogen-binding capacity. The rate and degree of clot contraction were significantly reduced in SLE patients compared with healthy subjects, especially in the patients with higher blood levels of anti-dsDNA antibodies. The reduced platelet contractility correlated with partial refractoriness of platelets isolated from the blood of SLE patients to stimulation induced by the thrombin receptor activating peptide. To test if the anti-dsDNA autoantibodies cause continuous platelet activation, followed by exhaustion and dysfunction of the cells, we added purified exogenous anti-dsDNA autoantibodies from SLE patients to normal blood before clotting. In support of this hypothesis, the antibodies first enhanced clot contraction and then suppressed it in a time-dependent manner. Importantly, a direct correlation of clot contraction parameters with the disease severity suggests that the reduced compactness of intravascular clots and thrombi could be a pathogenic factor in SLE that may exaggerate the impaired blood flow at the site of thrombosis. In conclusion, autoantibodies in SLE can affect platelet contractility, resulting in reduced ability of clots and thrombi to shrink in volume, which increases vessel obstruction and may aggravate the course and outcomes of thrombotic complications in SLE.

Factor XIII in plasma, but not in platelets, mediates red blood cell retention in clots and venous thrombus size in mice

AbstractThe transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIIIplasma) as a heterotetramer of A2 and B2 subunits and platelets (FXIIIplt) as an A2 homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIIIplasma, but not FXIIIplt, produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIIIplasma produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.

Reduced Contraction of Blood Clots in Venous Thromboembolism Is a Potential Thrombogenic and Embologenic Mechanism.

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.

Factor XIII cotreatment with hemostatic agents in hemophilia A increases fibrin α‐chain crosslinking

Background Hemophilia A results from the absence, deficiency or inhibition of factor VIII. Bleeding is treated with hemostatic agents (FVIII, recombinant activated FVII [rFVIIa], anti-inhibitor coagulation complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment, some patients have prolonged bleeding. FXIII-A2 B2 (FXIII) is a protransglutaminase. During clot contraction, thrombin-activated FXIII (FXIIIa) crosslinks fibrin and α2 -antiplasmin, which promotes red blood cell retention and increases clot stability and weight. We hypothesized that FXIII cotreatment in hemophilia would accelerate FXIII activation, leading to increased fibrin crosslinking. Methods FVIII-deficient plasma and whole blood were clotted with or without hemostatic agents (FVIII, rFVIIa, FEIBA, or recombinant B-domain-deleted porcine FVIII [rpFVIII]) and/or FXIII. The effects on FXIII activation, thrombin generation, fibrin and α2 -antiplasmin crosslinking, clot formation and clot weight were measured by western blotting, calibrated automated thrombography, thromboelastography, and clot contraction assays. Results As compared with FVIII-treated hemophilic plasma, FVIII + FXIII cotreatment accelerated FXIIIa formation without increasing thrombin generation. As compared with buffer-treated or FXIII-treated hemophilic plasma, FVIII treatment and FVIII + FXIII cotreatment increased the generation and amount of crosslinked fibrin, including α-chain-rich high molecular weight species and crosslinked α2 -antiplasmin. In the presence of FVIII inhibitors, as compared with hemostatic treatments (rFVIIa, FEIBA, or rpFVIII) alone, FXIII cotreatment increased whole blood clot weight. Conclusion In hemophilia A plasma and whole blood, FXIII cotreatment with hemostatic agents accelerated FXIIIa formation, increased the generation and amount of fibrin α-chain crosslinked species, accelerated α2 -antiplasmin crosslinking, and increased clot weight. FXIII cotreatment with hemostatic therapy may augment hemostasis through increased crosslinking of fibrin and α2 -antiplasmin.

Morphological Signs of Intravital Contraction (Retraction) of Pulmonary Thrombotic Emboli

The goal of the study was to establish whether contraction (retraction) of thrombi and/or thrombotic emboli occurs in vivo using morphological signs of blood clot compression, such as characteristic deformation of erythrocytes and redistribution of fibrin toward the surface of a thrombus/embolus. Three postmortem human pulmonary thrombotic emboli were examined by scanning electron microscopy and light microscopy after staining with hematoxylin and eosin as well as with Mallory’s stain. In two pulmonary emboli, which were extracted at 7 and 15 h after death of the patients, compressed polyhedral erythrocytes (polyhedrocytes) were revealed that were formed due to mechanical deformation under the action of contractile forces generated by activated platelets. In addition, the uneven distribution of fibrin within the emboli was found with accumulation of fibrin at the periphery of the emboli, which is another structural characteristic of a contracted blood clot. In one of the three emboli analyzed, which was extracted 38 h after the patient’s death (the “oldest” embolus), the morphological signs of contraction were absent, which was likely related to the partial postmortem autolysis of the embolus or intravital impairment of contraction. The ex vivo thrombotic emboli have morphological signs of clot contraction, suggesting intravital compression of the primary thrombi and/or thrombotic emboli. The in vivo contraction of thrombi and emboli may be an important pathogenic mechanism for modulation of blood flow past otherwise obstructive clots at the sites of thrombotic occlusion of a vessel. The presence of compressed erythrocytes inside and the predominant location of fibrin around the periphery of a thrombus or embolus can potentially serve as additional pathomorphological criteria for the intravital contraction of thrombi and thrombotic emboli.

Quantitative structural mechanobiology of platelet-driven blood clot contraction

Blood clot contraction plays an important role in prevention of bleeding and in thrombotic disorders. Here, we unveil and quantify the structural mechanisms of clot contraction at the level of single platelets. A key elementary step of contraction is sequential extension–retraction of platelet filopodia attached to fibrin fibers. In contrast to other cell–matrix systems in which cells migrate along fibers, the “hand-over-hand” longitudinal pulling causes shortening and bending of platelet-attached fibers, resulting in formation of fiber kinks. When attached to multiple fibers, platelets densify the fibrin network by pulling on fibers transversely to their longitudinal axes. Single platelets and aggregates use actomyosin contractile machinery and integrin-mediated adhesion to remodel the extracellular matrix, inducing compaction of fibrin into bundled agglomerates tightly associated with activated platelets. The revealed platelet-driven mechanisms of blood clot contraction demonstrate an important new biological application of cell motility principles.

Autoantibodies Against dsDNA Modulate Contraction of Blood Clots

The degree and rate of clot contraction (retraction) in systemic lupus erythematosus (SLE) patients, especially in those with a high level of anti-double stranded DNA (dsDNA) antibodies in the blood, was significantly reduced compared to healthy donors. We hypothesized that this effect was caused by the anti-dsDNA antibodies. To test this assumption, we investigated the kinetics of blood clot contraction in vitro in the absence and presence of anti-dsDNA antibodies purified from the blood serum of SLE patients. The degree of clot contraction was increased immediately after addition of the anti-DNA antibodies in a concentration-dependent manner. This stimulating effect was abrogated by a monoclonal antibody against the platelet Fc-receptor. On the contrary, after prolonged incubation (for hours) of the blood samples with the anti-DNA antibodies, the extent of clot contraction was significantly reduced. These results suggest that anti-dsDNA antibodies in SLE induce Fc-receptor-mediated chronic platelet hyperactivation, resulting in platelet exhaustion and dysfunction, including reduced contractility. The impaired contraction of blood clots and thrombi caused by autoantibodies may be an important pathogenic mechanism that affects the course and outcomes of thrombotic complications in SLE.

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis.

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions.

Activated Monocytes Enhance Platelet-Driven Contraction of Blood Clots via Tissue Factor Expression

Platelet-driven reduction in blood clot volume (clot contraction or retraction) has been implicated to play a role in hemostasis and thrombosis. Although these processes are often linked with inflammation, the role of inflammatory cells in contraction of blood clots and thrombi has not been investigated. The aim of this work was to study the influence of activated monocytes on clot contraction. The effects of monocytes were evaluated using a quantitative optical tracking methodology to follow volume changes in a blood clot formed in vitro. When a physiologically relevant number of isolated human monocytes pre-activated with phorbol-12-myristate-13-acetate (PMA) were added back into whole blood, the extent and rate of clot contraction were increased compared to addition of non-activated cells. Inhibition of tissue factor expression or its inactivation on the surface of PMA-treated monocytes reduced the extent and rate of clot contraction back to control levels with non-activated monocytes. On the contrary, addition of tissue factor enhanced clot contraction, mimicking the effects of tissue factor expressed on the activated monocytes. These data suggest that the inflammatory cells through their expression of tissue factor can directly affect hemostasis and thrombosis by modulating the size and density of intra- and extravascular clots and thrombi.

Interplay of Platelet Contractility and Elasticity of Fibrin/Erythrocytes in Blood Clot Retraction

Blood clot contraction (retraction) is driven by platelet-generated forces propagated by the fibrin network and results in clot shrinkage and deformation of erythrocytes. To elucidate the mechanical nature of this process, we developed a model that combines an active contractile motor element with passive viscoelastic elements. Despite its importance for thrombosis and wound healing, clot contraction is poorly understood. This model predicts how clot contraction occurs due to active contractile platelets interacting with a viscoelastic material, rather than to the poroelastic nature of fibrin, and explains the observed dynamics of clot size, ultrastructure, and measured forces. Mechanically passive erythrocytes and fibrin are present in series and parallel to active contractile cells. This mechanical interplay induces compressive and tensile resistance, resulting in increased contractile force and a reduced extent of contraction in the presence of erythrocytes. This experimentally validated model provides the fundamental mechanical basis for understanding contraction of blood clots.

Role of red blood cells in haemostasis and thrombosis.

In contrast to an obsolete notion that erythrocytes, or red blood cells (RBCs), play a passive and minor role in hemostasis and thrombosis, over the past decades there has been increasing evidence that RBCs have biologically and clinically important functions in blood clotting and its disorders. This review summarizes the main mechanisms that underlie the involvement of RBCs in hemostasis and thrombosis in vivo, such as rheological effects on blood viscosity and platelet margination, aggregation and deformability of RBCs; direct adhesion and indirect biochemical interactions with endothelial cells and platelets, etc. The ability of stored and pathologically altered RBCs to generate thrombin through exposure of phosphatidylserine has been emphasized. The procoagulant and prothrombotic potential of RBC-derived microparticles transfused with stored RBCs or formed in various pathological conditions associated with hemolysis has been described along with prothrombotic effects of free hemoglobin and heme. Binding of fibrinogen or fibrin to RBCs may influence their effects on fibrin network structure, clot mechanical properties, and fibrinolytic resistance. Recent data on platelet-driven clot contraction show that RBCs compressed by platelets pulling on fibrin form a tightly packed array of polyhedral erythrocytes, or polyhedrocytes, which comprises a nearly impermeable barrier important for hemostasis and wound healing. RBCs may perform dual roles, both helping to stem bleeding but at the same time contributing to thrombosis in a variety of ways.

Blood Clot Contraction (Retraction) Is Impaired in Acute Ischemic Stroke

Obstructive thrombi or thrombotic emboli of cerebral arteries are the pathogenic basis of ischemic stroke, which is a leading cause of death and disability worldwide. Blood clots undergo volume shrinkage due to the contractile forces that are generated by platelets and propagated through the clot volume due to platelet-fibrin interactions and elasticity of the fibrin network. This process is designed clot contraction (retraction) which remains one of the least studied steps of blood clotting. Importantly, this phenomenon has been shown to occur not only in vitro but also in in vivo thrombi. Clot contraction has been shown to be important in the volume reduction of otherwise obstructive thrombi and has the potential to reduce occlusion and restore blood flow past emboli or thrombi. Despite the potential medical significance of clot contraction, it has not been examined systemically in a clinical setting. This aim of this work was to examine the potential pathogenic role of clot contraction in ischemic stroke. Here we employed a novel automated method to quantify the time of initiation, extent and rate of clot contraction in vitro to compare clot contraction in the blood of healthy subjects with patients suffering a recent ischemic stroke (<6 hours from the onset of symptoms) who had not yet received any treatment with anticoagulants, antiplatelet drugs or thrombolytics.. Parameters of clot contraction were correlated with the severity and etiological types of stroke as well as with hematological, coagulation, and biochemical tests to examine the clinical significance of clot contraction. The main finding of this work is that clot contraction in blood from patients with acute ischemic stroke is reduced on average by ~60% (p<0.0001) when compared to that of healthy subjects. The reduction in clot contraction is correlated with a lower platelet count and platelet dysfunction, higher fibrinogen level, higher hematocrit, leukocytosis as well as other changes in the blood composition of patients with ischemic stroke that may alter the properties of the blood clot. We propose that these changes in the composition of the blood contribute to the impaired mechanism of clot contraction, which may exaggerate vessel occlusion and brain infarct. While stroke severity is determined mainly by the diameter and location of the obstructed cerebral artery, the ability of the thrombi to contract more or less may augment or ease the course of brain damage. Clinical correlations with respect to severity and stroke etiology indicate that reduced clot contraction has the potential to be a pathogenic factor in ischemic stroke. Paradoxically, the extent of clot contraction marginally improved in patients with a more severe stroke (NIHSS>15 vs. NIHSS<15, p<0.01), while it was still significantly reduced compared to healthy subjects. This finding can be presumably explained by the fact that in severe brain damage a massive amount of tissue factor is released into the systemic circulation, which can induce the activation of blood coagulation. We propose that this release of tissue factor results in a secondary wave of thrombin generation that causes patients with more severe stroke to have hyperactivity of platelets. In combination with a higher platelet count (p<0.01) this can enhance contraction of obstructive clots or thrombi, which may be a compensatory mechanism resulting in the recanalization of an otherwise occluded blood vessel. In support of this hypothetical scenario, it was also found that patients with atherothrombotic strokes have an increased extent of clot contraction compared to patients with cardioembolic stroke (p<0.05), and atherothrombotic patients are reported to have increased tissue factor as a consequence of atherosclerotic lesions. In summary, the clinical pathophysiological importance of clot contraction in a thrombotic state has been examined for the first time and the modulation of the ability of clots or thrombi to shrink in volume may be a novel and unappreciated mechanism that aggravates or alleviates the course and outcomes of thrombosis, such as ischemic stroke. The clinical importance of clot or thrombus remodeling in vivo as well as the diagnostic and prognostic value of this blood test for clot contraction needs further exploration. DisclosuresWeisel:Bayer: Research Funding.