Incipient Clot Research Articles

Exercise transiently increases the density of incipient blood clots in antiplatelet-treated lacunar stroke patients

IntroductionOlder individuals and, in particular, individuals at risk of recurrent stroke, may be susceptible to thrombosis when participating in exercise, however, this aspect has not been well investigated.MethodsClot microstructure and conventional markers of thrombotic risk were determined in twenty lacunar stroke patients and fifteen healthy age-matched controls before, immediately after and 1 h after a bout of moderate intensity cycling exercise. Data were analyzed using a linear mixed model approach.ResultsAt rest, clot microstructure (1.69 ± 0.07 vs. 1.64 ± 0.05, corresponding to a difference of ~ 50% in normalized clot mass; p = 0.009) and thrombocyte count (73%; p < 0.0001) were higher, and activated partial thromboplastin time was lower (18%; p = 0.0001) in stroke patients compared to age-matched controls. Acute exercise increased thrombogenic markers similarly in the two groups: incipient clot microstructure (1.69 ± 0.07 vs. 1.74 ± 0.05; p = 0.0004 and 1.64 ± 0.05 vs. 1.71 ± 0.04; p < 0.0001, for stroke and controls respectively), plasma fibrinogen (12%; p < 0.0001 and 18%; p < 0.0001, for stroke and controls respectively) and the combined coagulation factors II, VII and X (p = 0.0001 and p < 0.0001, for stroke and controls respectively).ConclusionThe results show that exercise transiently increases the risk of blood clot formation in both stroke patients and controls, however, due to the higher baseline thrombogenicity in stroke patients, the post exercise risk of forming blood clots may be higher in this group.Trial registrationRegistered at ClinicalTrials.gov (NCT03635177).

Exercise training induces thrombogenic benefits in recent but not late postmenopausal females.

Although regular physical activity is known to improve cardiovascular health in men, evidence for its beneficial effects in postmenopausal females is less convincing and it remains unclear whether initiation of exercise training soon after, rather than many years after menopause impacts the magnitude of training-induced adaptations. We evaluated exercise-induced changes in markers of thrombotic risk and conduit artery function in recent≤5yr compared with late≥10yr postmenopausal females. Fourteen recent≤5yr and 13 late≥10yr healthy postmenopausal females completed 8 wk of regular intensive exercise training, consisting of floorball and cycling. Markers of thrombotic risk and vascular health were assessed before and after the intervention, and data were analyzed using a linear mixed model. Exercise training reduced markers of thrombotic risk, including an 11% reduction (P = 0.007) in agonist-induced platelet reactivity and a reduction (P = 0.027) in incipient clot microstructure (∼40% reduction in clot mass) in the recent≤5yr but not the late≥10yr (P = 0.380; P = 0.739, respectively) postmenopausal females. There was no change in conduit artery function, as measured by brachial (recent≤5yr, P = 0.804; late≥10yr, P = 0.311) and popliteal artery (recent≤5yr, P = 0.130; late≥10yr, P = 0.434) flow-mediated dilation. Only the late≥10yr postmenopausal females exhibited an increase (by 9.6%, P = 0.022) in intracellular adhesion molecule-1 levels after training, which may have impacted the thrombogenic adaptation in this group. These findings suggest that 8 wk of high-intensity exercise training reduces thrombotic risk in recent≤5yr, but not late≥10yr postmenopausal females. Thus, regular physical activity initiated soon after, rather than many years after menopause and at a higher age, may be more efficient for reducing thrombogenic risk.NEW & NOTEWORTHY Eight weeks of high-intensity exercise training reduces platelet reactivity as well as blood clot density and strength in females ≤5 yr past menopause but not in females ≥10 yr past menopause. The divergent response in the late postmenopausal females may be explained by training-induced low-grade systemic inflammation. These findings suggest that regular physical activity initiated soon after menopause, compared with many years after menopause, may be more efficient for reducing the risk of blood clots.

The efficacy of low molecular weight heparin is reduced in COVID-19.

A significant degree of mortality and morbidity in COVID-19 is through thromboembolic complications, only partially mitigated by anticoagulant therapy. Reliable markers of infection severity are not fully established. This study investigated whether visco-elastic biomarkers predict disease severity on presentation to the Emergency Department (ED) and how they measure response to anticoagulationMETHODS:Patients testing positive for COVID-19 at a large University Teaching Hospital ED were recruited at presentation. Multiple blood samples were taken throughout hospital admission to monitor disease progression with end outcome recorded. Visco-elastic markers, fractal dimension (df) and Time to Gel Point (TGP) which measure the properties of the incipient clot were compared in patients with and without anticoagulation by Low Molecular Weight Heparin (LMWH). TGP and df did not predict severity of infection with COVID-19. Although LMWH prolonged TGP, there was no change in df indicating LMWH did not change clot microstructure. Therapeutic efficacy of LMWH appears blunted in COVID-19 infection. This may be due to the inflammatory state creating a resistance to LMWH activity, which may explain why LMWH appears less effective in COVID-19 compared to other disease states. COVID-19 was not predicted by visco-elastic testing at the time of ED presentation.

The treatment effect of rivaroxaban on clot characteristics in patients who present acutely with first time deep vein thrombosis.

The acute vascular disease deep vein thrombosis (DVT) requires oral anticoagulants to prevent progression. Monitoring therapeutic efficacy of direct oral anticoagulants (DOAC), including rivaroxaban, is problematic as no reliable test is available. Advances in rheometry have led to the development of a functional coagulation biomarker using Gel Point (GP) analysis which assesses clot structure formation. The biomarker measures incipient clot formation time (TGP) and quantifies fibrin clot structure in terms of fractal dimension (df). This study aimed to investigate clot structure formation in first time DVT and the effect of rivaroxaban treatment. This prospective observational cohort study measured the GP and standard laboratory markers at three sample points: pre-treatment and at 20 and 60 days following 15 mg BD and 20 mg OD rivaroxaban respectively. Forty DVT patients (mean age 64 years [SD±14.8]; 23 males, 17 female) were recruited. The results show that DVT vs non-DVT patients did not have a significantly different GP profile (df: 1.72±0.06 vs 1.70±0.06 and TGP: 267±68 sec vs 262±73 sec) with both within the defined healthy index. In addition, rivaroxaban therapy increased TGP to 392 s (±135 s) after 20 days, and subsequently increased to 395 s (±194 s) at 60 days but did not significantly increase df (from 1.69±0.05 to 1.71±0.06). The results indicate in this cohort of DVT patients there was no underlying hypercoagulable effect as determined by gel point analysis. Furthermore, the anticoagulant effect of rivaroxaban prolonged clotting, suggesting a protective effect against clot formation, without significantly reducing clot microstructural properties.

In vitro clot model to evaluate fibrin-thrombin effects on fractal dimension of incipient blood clot.

This aim of this study is to investigate the individual effects of varying concentrations of thrombin and fibrinogen on clot microstructure (characterised through the fractal dimension of the incipient clot network, df) and clot formation time (TGP) using a fibrin-thrombin clot model. df and TGP markers are measured using a haemorheological method that has already been investigated for whole blood. This is an in vitro study using three thrombin concentrations (0.1, 0.05 and 0.02 NIH/ml) and two fibrinogen concentrations (8 mg/ml and 12 mg/ml) to investigate a fibrin-thrombin clot model. The haemorheological changes were measured at the gel point using df and TGP. Fractal dimension (df) increased with increasing concentrations of thrombin both at 8 mg/ml (1.60±0.024, 1.67±0.022, 1.74±0.079) and 12 mg/ml fibrinogen concentrations (1.63±0.02, 1.87±0.019, 1.95±0.014). On the other hand, TGP decreased for both 8 mg/ml (1089±265, 637±80, 223±22 seconds) and 12 mg/ml fibrinogen concentrations (2008±247, 776±20, 410±20 seconds). In contrast to previous studies investigating whole blood, TGP increased with higher fibrinogen levels. The findings from this fibrin-thrombin clot model confirmed that df and TGP can detect changes in the incipient clot following manipulation of fibrinogen and thrombin concentration. df increases (indicating stronger clot) with higher concentrations of thrombin and fibrinogen. On the other hand, TGP decreased as expected with higher thrombin level but not with higher fibrinogen concentrations.

Effects of unidirectional flow shear stresses on the formation, fractal microstructure and rigidity of incipient whole blood clots and fibrin gels.

Incipient clot formation in whole blood and fibrin gels was studied by the rheometric techniques of controlled stress parallel superposition (CSPS) and small amplitude oscillatory shear (SAOS). The effects of unidirectional shear stress on incipient clot microstructure, formation kinetics and elasticity are reported in terms of the fractal dimension (df) of the fibrin network, the gel network formation time (TGP) and the shear elastic modulus, respectively. The results of this first haemorheological application of CSPS reveal the marked sensitivity of incipient clot microstructure to physiologically relevant levels of shear stress, these being an order of magnitude lower than have previously been studied by SAOS. CSPS tests revealed that exposure of forming clots to increasing levels of shear stress produces a corresponding elevation in df, consistent with the formation of tighter, more compact clot microstructures under unidirectional flow. A corresponding increase in shear elasticity was recorded. The scaling relationship established between shear elasticity and df for fibrin clots and whole blood confirms the fibrin network as the dominant microstructural component of the incipient clot in terms of its response to imposed stress. Supplementary studies of fibrin clot formation by rheometry and microscopy revealed the substantial additional network mass required to increase df and provide evidence to support the hypothesis that microstructural changes in blood clotted under unidirectional shear may be attributed to flow enhanced thrombin generation and activation. CSPS also identified a threshold value of unidirectional shear stress above which no incipient clot formation could be detected. CSPS was shown to be a valuable haemorheological tool for the study of the effects of physiological and pathological levels of shear on clot properties.

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution

IntroductionWe investigated the effect of progressive haemodilution on the dynamics of fibrin clot formation and clot microstructure using a novel rheological method. The technique measures clotting time (TGP), clot strength (G`GP), and quantifies clot microstructure (df) at the incipient stages of fibrin formation. We use computational modelling to examine the relationship between structure and mass, as well as helium ion microscopy (HIM) to compare morphological changes in the fully formed clot to that of the incipient clot. MethodsThis is an in vitro study; 90 healthy volunteers were recruited with informed consent and a 20ml sample of whole blood obtained from each volunteer. Five clinically relevant dilutions were investigated using 0.9w.v isotonic saline (0, 10, 20, 40 and 60%, n=18 for each dilution). The rheological method of assessing structural clot changes was compared against conventional coagulation screen and fibrinogen estimation. ResultsFractal dimension (df) and final clot microstructure both decreased with progressive dilution (significant at a dilution of 20%) with similar relationships observed for final clot characteristics in HIM images. Significant correlations were observed between df and G`GP (clot strength) (0.345, p=0.02), as well as clotting time (PT: −0.690, p>0.001; APTT: −0.672, p>0.001; TGP: −0.385, p=0.006). ConclusionsThis study provides new insight into the effects of haemodilution by isotonic saline on clotting time (TGP), clot strength (G′GP) and clot microstructure (df). Previous studies have attempted to link clot microstructure to clot quality/strength, however this study provides a significant step in quantifying these relationships.

P256 Treatment of obstructive sleep apnoea syndrome with continuous positive airways pressure alters haemostasis: further data on the use of fractal analysis to measure microstructure of incipient clot

IntroductionUntreated obstructive sleep apnoea syndrome (OSAS) increases cardiovascular risk and altered haemostasis is at least partly implicated. As previously reported using fractal analysis and a new biomarker called fractal dimension...

S43 Fractal analysis of whole blood incipient clot confirms an early morning prothrombotic state in patients with untreated Obstructive Sleep Apnoea Syndrome

IntroductionObstructive sleep apnoea syndrome (OSAS) independently increases cardiovascular risk and a pro-thrombotic state has been at least partly implicated. Using fractal analysis we have developed a new biomarker called fractal...

Fractal discrimination of random fractal aggregates and its application in biomarker analysis for blood coagulation

A recent rheological study has established that the fractal dimension, df, of an incipient clot, formed at the Gel Point (sol–gel transition) of coagulating blood is a significant new biomarker of haemostasis. In whole healthy blood, incipient clots show a clearly defined value of df=1.7 within a narrow range, which represents a new ‘healthy index’ for normal clotting. The addition of unfractionated heparin significantly prolongs the onset of clot formation with a corresponding reduction of df as a function of heparin dose. However, as clots mature they exhibit (i) an expected increase in df and (ii) a significant increase to spread of these values, i.e. df’s in the range 2.0–2.5, limiting the use of df as a discriminant of clot microstructure.The present study, details how and why the spectral dimension, ds, can be used to accommodate this shortcoming and allow discrimination of mature forms of clot microstructure in indistinguishable in terms of their fractal dimension. To elucidate why ds permits discrimination a numerical experiment was conducted on computationally generated random fractal aggregates (RFAs) with a priori set value of df. Starting from RFAs with a df of 1.7, mature RFAs are evolved from these incipient templates by two differing growth processes achieving a final df of 2.1. Fractal and statistical analysis of the mature RFAs reveals, for the first time, that their differing internal structure is manifest in the magnitude of ds. The potential clinical significance of these findings is discussed in terms of the possibility of exploiting the incipient clot’s ability to template the internal arrangement of the mature clot to better predict long term clot susceptibility to lysis.

Rheometrical and molecular dynamics simulation studies of incipient clot formation in fibrin-thrombin gels: An activation limited aggregation approach

A rheometrical investigation of incipient clots formed in fibrin-thrombin gels is reported in which the Gel Point (GP) is characterised by frequency independence of the loss tangent in small amplitude oscillatory shear measurements over a wide range of thrombin concentration. Values of the fractal dimension ( D f ) of the GP network calculated from measurements are consistent with those reported in simulations of diffusion limited cluster–cluster aggregation (DLCCA) and reaction limited cluster–cluster aggregation (RLCCA), but differ insofar as the values of D f calculated from the present experiments increase progressively with a reduction in gel formation time. A molecular dynamics simulation (MDS) of systems of rod-like particles was designed to (i) test the hypothesis that the presence of an activation profile in a cluster aggregation model could account for the trend of D f as a function of gel formation time observed experimentally in fibrin-thrombin gels and whole heparinised blood without recourse to the inclusion of fibrinogen-specific interactions; and (ii) to explore the effect of monomer activation kinetics on the microstructure of fractal clusters formed in systems of rigid rod-like particles. The results identify two possible mechanisms for the increase in D f as the gel formation time decreases, both being a consequence of altering the evolution of the clustering dynamics by a process referred to herein as activation limited aggregation (ALA). This ALA-based MDS substantiates the experimental findings by confirming the trend evident in the formation of incipient clots in fibrin-thrombin gels and in whole heparinised blood. A mechanism for ALA involving the aggregation of pre-GP sub-clusters is proposed.

Gel point and fractal microstructure of incipient blood clots are significant new markers of hemostasis for healthy and anticoagulated blood

AbstractHere we report the first application of a fractal analysis of the viscoelastic properties of incipient blood clots. We sought to ascertain whether the incipient clot's fractal dimension, Df, could be used as a functional biomarker of hemostasis. The incipient clot is formed at the gel point (GP) of coagulating blood, the GP demarcating a functional change from viscoelastic liquid to a viscoelastic solid. Incipient clots formed in whole healthy blood show a clearly defined value of Df within a narrow range that represents an index of clotting in health, where Df = 1.74 (± 0.07). A significant relationship is found between the incipient clot formation time, TGP, and the activated partial thromboplastin time, whereas the association of Df with the microstructural characteristics of the incipient clot is supported by its significant correlation with fibrinogen. Our study reveals that unfractionated heparin not only prolongs the onset of clot formation but has a significant effect on its fractal microstructure. A progressive increase in unfractionated heparin concentration results in a linear decrease in Df and a corresponding prolongation in TGP. The results represent a new, quantitative measure of clot quality derived from measurements on whole blood samples.

The development of rheometry for strain-sensitive gelling systems and its application in a study of fibrin–thrombin gel formation

This paper reports simulated sequential frequency sweep data which have been reconstructed from time resolved viscoelastic data obtained by Fourier transform mechanical spectroscopy. Comparisons of the results show that the recording of anomalous values of the stress relaxation power law exponent α at the Gel Point under ‘rapid’ gelling conditions may be due to inappropriate rheological techniques. An appropriate rheometrical criterion is established for the application of sequential frequency sweeps in order to obtain accurate values of α in the formation of strain-sensitive, rapidly formed gels. Furthermore, using appropriate rheometry, we report values of α for fibrin–thrombin gels formed by the addition of thrombin to a physiologically relevant level of human fibrinogen, and relate these values to the microstructure of the fibrin gel network in terms of a fractal dimension. The present study is the first to report a modification of the fractal characteristics of incipient clots in fibrin–thrombin gels due to the availability of thrombin. This work confirms the hypothesis that the self-similar (fractal) stress relaxation behaviour recorded at the Gel Point of samples of coagulating blood (Evans et al. 2010a, b) is associated with the microstructural characteristics of the incipient blood clot’s fibrin network.

Gel point and fractal microstructure of incipient blood clots are significant new markers of haemostasis

We have investigated the hypothesis that, in whole blood, the incipient clot formed at the gel point (GP) is characterised by a fractal microstructure1 2 and that this could be...

Gel point and fractal microstructure of incipient blood clots are significant new markers of haemostasis

We have investigated the hypothesis that in whole blood the incipient clot formed at the gel point (GP) is characterised by a fractal microstructure [1,2] and that this could be detected in healthy and anticoagulated blood. We compared the gel time (GT) required to form the incipient clot and the corresponding fractal dimension (df) against laboratory markers of haemostasis and thromboelastography (TEG).

Rheometrical detection of incipient blood clot formation by Fourier transform mechanical spectroscopy

We report a study of the coagulation of whole blood by oscillatory shear Fourier transform mechanical spectroscopy. The results include the first identification of the Gel Point of coagulating blood in terms of the Chambon–Winter Gel Point criterion and we show how this may be used as an appropriate basis for detecting the establishment of an incipient clot in samples of human blood. A comparison of this technique with results obtained from a thromboelastograph (TEG) indicate that the latter is not capable of detecting the incipient clot, whose establishment can occur several minutes prior to the first recorded TEG response. Our results also reveal, for the first time, the incipient clot's fractal characteristics. An analysis of the viscoelastic data obtained at the Gel Point of samples of whole blood drawn from healthy individuals indicate that the incipient clot is characterised by a fractal dimension d f = 1.74(±0.07). The significance of these findings is discussed in terms of their potential application in studies of pathologically induced changes in blood clot microstructure.

Interactions of fibrinolytic system proteins with lysine-containing surfaces.

Studies on the interactions of tissue plasminogen activator (tPA) and plasminogen with polyurethane surfaces containing epsilon-lysine moieties (epsilon-amino group free) are reported. These surfaces are considered to have the potential to dissolve nascent clots that may be formed on them. For adsorption from both single protein solutions and plasma, the surfaces were found to have a high capacity for tPA as well as plasminogen. A significant fraction of preadsorbed tPA was displaced from the epsilon-lysine surfaces upon contact with plasma. These surfaces, when preadsorbed with tPA and then incubated with plasma, were able to dissolve incipient clots formed around them. However, the clot-dissolving capacity diminished as the time of plasma incubation increased, presumably due to loss of tPA. It was also shown that in plasma, preadsorbed tPA is displaced from these surfaces largely by plasminogen, which thus appears to have a greater binding affinity than tPA for the epsilon-lysine moieties. Finally, it was found that in plasma, the epsilon-lysine surfaces interact with plasminogen in a dynamic manner, and that about 70% of the bound plasminogen is exchanging continuously with plasminogen in the plasma.

Lysine-derivatized polyurethane as a clot lysing surface: conversion of adsorbed plasminogen to plasmin and clot lysis in vitro

Polyurethane surfaces to which lysine residues are immobilized by photochemical methods are proposed as a basis for clot lysing surfaces. The lysines are attached in such a way that the ε-amino and carboxyl groups are free. We showed previously that these surfaces, when placed in contact with plasma, adsorb only plasminogen and virtually no other proteins (McClung et al., J. Biomed. Mater. Res. 49 (2000) 409). In this communication, data based on a chromogenic substrate assay are presented showing that plasminogen adsorbed to these surfaces is readily converted to plasmin in the presence of tissue-plasminogen activator (t-PA). Moreover, the rate of activation on the surface is considerably greater than in solution. Experiments demonstrating the ability of these surfaces to dissolve fibrin clots are also reported. Surfaces exposed to plasma and then to t-PA were placed in citrated plasma. On recalcification, clotting was initiated, but the incipient clots were soon dissolved. On control surfaces (no lysine or lysine in which the ε-amino groups were not available) coagulation continued until a stable clot was formed. Similar observations were made when the plasma/t-PA exposed surfaces were placed in a pure fibrinogen solution and thrombin was added.

Stirring the blood

Blood clotting in the wrong place in the human body can be a killer. Daedalus plans to tackle the problem with an innovative use of ultrasound. A converging beam directed for a time to, say, one of the wider arteries, will agitate the blood, break up any incipient clots and keep blood safely fluid.