Stages Of Thrombus Formation Research Articles

Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of shear rate gradients (SRG) but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create SRG during blood perfusion. VWF-GPIbα interactions were increased at sites of SRG compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRG but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFVA1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRG on VWF dependent platelet aggregation and developed the shear gradient-sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of SRG. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal hemostasis.

Rapid effects of aldosterone on platelets, coagulation, and fibrinolysis lead to experimental thrombosis augmentation

An increase in aldosterone levels positively correlates with an increased risk of acute cardiovascular thrombotic events. The aim of the study was to determine the mechanism of action of prothrombotic aldosterone focusing on the rapid effects of the hormone on platelets, coagulation, and fibrinolysis. A wide panel of advanced ex vivo and in vitro techniques was used for the evaluation of coagulation and fibrinolysis in aldosterone-treated rats. Additionally, two experimental mice models of thrombosis, which allowed for the intravital observation of the first stage of thrombus formation in real time, were used. Acute administration of aldosterone in rats increased the density of fibrin net and platelet aggregates in clots as well as reduced fibrinolysis. These effects were observed within 10 min and were partially suppressed by eplerenone. Moreover, acute administration of aldosterone in mice enhanced platelet accumulation at the site of endothelial injury induced by laser and increased the area of irreversibly activated platelets in FeCl3-induced thrombus. These results demonstrate that aldosterone acutely affects platelets, coagulation, and fibrinolysis, leading to an enhanced thrombosis. The aldosterone effects were mediated partially via a mineralocorticoid receptor. The mechanism seems to involve non-genomic signaling since the effects were observed within a few minutes of aldosterone administration.

Thrombus Formation at High Shear Rates.

The final common pathway in myocardial infarction and ischemic stroke is occlusion of blood flow from a thrombus forming under high shear rates in arteries. A high-shear thrombus forms rapidly and is distinct from the slow formation of coagulation that occurs in stagnant blood. Thrombosis at high shear rates depends primarily on the long protein von Willebrand factor (vWF) and platelets, with hemodynamics playing an important role in each stage of thrombus formation, including vWF binding, platelet adhesion, platelet activation, and rapid thrombus growth. The prediction of high-shear thrombosis is a major area of biofluid mechanics in which point-of-care testing and computational modeling are promising future directions for clinically relevant research. Further research in this area will enable identification of patients at high risk for arterial thrombosis, improve prevention and treatment based on shear-dependent biological mechanisms, and improve blood-contacting device design to reduce thrombosis risk.

Free Fatty Acids Modulate Thrombin Mediated Fibrin Generation Resulting in Less Stable Clots.

Upon platelet activation, free fatty acids are released at the stage of thrombus formation, but their effects on fibrin formation are largely unexplored. Our objective was to characterize the kinetic effects of fatty acids on thrombin activity, as well as the structural and mechanical properties of the resultant fibrin clots. Thrombin activity on fibrinogen was followed by turbidimetry and detailed kinetic characterization was performed using a fluorogenic short peptide substrate. The viscoelastic properties of fibrin were measured with rotatory oscillation rheometer, whereas its structure was analyzed with scanning electron microscopy (SEM). In turbidimetric assays of fibrin generation, oleate and stearate at physiologically relevant concentrations (60–600 μM) produced a bell-shaped inhibitory dose response, increasing 10- to 30-fold the time to half-maximal clotting. Oleate inhibited thrombin activity on a short peptide substrate according to a mixed-type inhibitor pattern (a 9-fold increase of the Michaelis constant, Km and a 20% decrease of the catalytic constant), whereas stearate resulted in only a minor (15%) drop in the catalytic constant without any change in the Km. Morphometric analysis of SEM images showed a 73% increase in the median fiber diameter in the presence of stearate and a 20% decrease in the presence of oleate. Concerning the viscoelastic parameters of the clots, storage and loss moduli, maximal viscosity and critical shear stress decreased by 32–65% in the presence of oleate or stearate, but loss tangent did not change indicating decreased rigidity, higher deformability and decreased internal resistance to shear stress. Our study provides evidence that free fatty acids (at concentrations comparable to those reported in thrombi) reduce the mechanical stability of fibrin through modulation of thrombin activity and the pattern of fibrin assembly.

Platelet glycoprotein gene Ia C807T, HPA-3, and Ibα VNTR polymorphisms are associated with increased ischemic stroke risk: Evidence from a comprehensive meta-analysis.

Background/aims Platelet glycoproteins play a crucial role in the initial stage of thrombus formation and may contribute to the pathophysiology of atherosclerosis. Polymorphisms in glycoprotein genes alter the function of the protein, possibly leading to increased risk of ischemic stroke. However, previous genetic association studies that examined the relationship between glycoprotein genes polymorphisms and ischemic stroke have yielded inconsistent results. This study aimed to evaluate the association between glycoprotein genes and ischemic stroke by the application of meta-analysis. Methods Relevant studies were identified by an extensive search through databases. The quality of included studies was assessed independently using the Newcastle-Ottawa Scale. Allele and genotype frequencies for each included study were extracted. The odds ratio (OR) with 95% confidence interval (95%CI) was calculated using a random-effects or fixed-effects model. Q statistic was used to evaluate homogeneity, and a meta-regression model was used to explore the study-level variables and to describe the heterogeneity in included studies. Egger's test and funnel plot were used to assess publication bias. Results A total of 60 studies (9 polymorphisms) were included and identified in the current meta-analysis. The Newcastle-Ottawa Scale scores ranged from 7 to 9 except for two studies with Newcastle-Ottawa Scale scores of 6. The T allele or TT genotype of the glycoprotein Ia C807T polymorphism were associated with an increased susceptibility to ischemic stroke in combined population (807T allele: OR, 95%CI: 1.24, 1.03-1.50, p = 0.02) or Asian populations (807T allele: OR, 95%CI: 1.31, 1.10-1.54, p = 0.002 and 807TT genotype: OR, 95%CI: 1.53, 1.13-2.08, p = 0.006, respectively), and the Ser allele of HPA-3 was associated with increased risk of ischemic stroke in combined population or in Asians (OR, 95%CI: 1.21, 1.04-1.40, p = 0.01 or 1.54, 1.18-2.01, p = 0.001). Of note, the Ser/Ser genotype was more common in Asians (OR, 95%CI: 2.09, 1.40-3.13, p < 0.001). For glycoprotein Ibα variable number tandem repeat, only B allele showed a mild significant association with ischemic stroke risk in combined population or in Caucasians (OR, 95%CI: 2.17, 1.04-4.55, p = 0.04 or 1.79, 1.02-3.13, p = 0.04). There was no significant association between HPA-1, HPA-2, HPA-4, HPA-5, glycoprotein Ibα-5 T/C as well as Ia G873A polymorphisms and increased risk of ischemic stroke. Conclusions We found that glycoprotein Ia C807T T allele or the TT genotype, the Ser-allele of HPA-3 and B allele of glycoprotein Ibα variable number tandem repeat polymorphisms were associated with increased risk for ischemic stroke. Future studies with larger sample sizes will be necessary to confirm the results. In addition, analyses of ischemic stroke subtypes and gene-gene and gene-environment interactions are warranted.

ACTIVATION OF PROTEIN C IN THE IN VITRO THROMBOLYSIS

Physiological conditions of formation and subsequent lysis of thrombus were reconstituted in vitro in our research. Thrombus formation was initiated either by addition of exogenous thrombin or by contact of blood with anionic surface, which stimulates spontaneous coagulation of blood. Tissue plasminogen activator and/or protein C were previously added in the blood sample. The time of the beginning and total degradation of formed thrombi as well as the level of PC in lysates was controlled then. Only an addition of protein C alone or in combination with tissue plasminogen activator led to the most effective lysis of thrombi: their residual weight was 18% and 5% comparing to control. Addition of exogenous tissue plasminogen activator alone or in combination with protein C caused a 83% and 74% decrease of PC level in lysates of spontaneously formed thrombi, and 72% and 56% decrease for thrombi formed by thrombin, respectively. Without an addition of tissue plasminogen activator protein C level in lysates of thrombi formed by thrombin was 54% down on spontaneously formed thrombi. Thus, changes of PC concentration in isolated volume of clot seem to be controlled by thrombin at the stage of thrombus formation and by fibrinolytic system at the stage of fibrinolysis. Concentration of PC in lysates from clots formed by exogenous thrombin was decreasing over the next 10 hours of thrombolysis, which can also be the evidence of the interaction between the fibrinolytic and PC activation systems. A hypothesis is. formulated about an existence of endothelium-independent mechanism of PC activation in blood plasma with blood cells participation, which effectiveness increases in the process of thrombolysis.

Thrombosis Therapy: Focus on Antiplatelet Agents

Platelet adhesion, activation and aggregation to the injured vessel wall are crucially involved in the pathogenesis of thrombus formation. Agents in theory thwarting these phases would have significant clinical value. The current antiplatelet drugs used in daily clinical practice include COX-1 inhibitor aspirin, ADP P2Y12 receptor antagonist clopidogrel, and the GPIIb-IIIa antagonists (abciximab, eptifibatide and tirofiban). However, confined curative ratio along with unforeseen bleeding risk remains a major puzzle of antiplatelet therapy. With advances in understanding of the molecular basis of platelet in thrombosis, newer antiplatelet agents that targets different stage of thrombus formation have been recently developed, mostly including agents targeting platelet adhesion (GPIV, vWF), activation (GPVI, P2Y12, TPα, PAR1, phosphodiesterase, cyclooxygenase), and aggregation (GPIIb/IIIa). In this article, we will review the advantages and limitations of various antiplatelet agents that have been approved by the US Food and Drug Administration (FDA) or under development.

Polyphosphates: a link between platelet activation, intrinsic coagulation and inflammation?

Evaluation of: Müller F, Mutch NJ, Schenk WA et al. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell 139, 1143–1156 (2009).Early evidence has already shown that activated platelets play a role in the intrinsic pathway of coagulation, but the mechanism remained unclear. The finding that the dense granules of platelets contain polyphosphate (polyP), which has procoagulant effects, resolved this missing link between activated platelets and coagulation. PolyP secreted from platelets leads via activation of Factor XII to in vivo thrombin and fibrin formation. The relative importance of polyP in the process and stages of thrombus formation will require further investigation. In addition to its role in coagulation, polyP-induced Factor XII activation mediates the release of the inflammatory mediator bradykinin by activating the kallikrein–kinin system. This results in vascular leakage and edema formation in vivo. Together, polyP release from platelets has a physiological role in the thrombus-forming process, as well as in inflammatory reactions.

Prevention of thromboembolism.

Recently much attention has been focused on the high incidence of thromboembolism following major hip surgery–and in particular total hip reconstruction. Mortality is at least 1.8% following total hip replacement in the absence of prophylaxis. Because of the difficulties encountered with use of anticoagulants such as warfarin sodium and heparin sodium, attention has been turned toward the dextran solutions as antiplatelet agents. Basic, however, to the use of an antiplatelet agent to prevent thromboembolism is an understanding of the central role of the platelet in thrombogenesis. Histopathologic studies have shown that the development of a mural platelet thrombus is the first stage of thrombus formation, not only in the arterial but also in the venous system. A number of studies have further shown that many patients who develop thromboemboli show an abnormality in platelet adhesiveness. The available evidence suggests that the activation of the venous thrombus may follow the formation