Clot Composition Research Articles

Fondue and transglutaminase in the Drosophila larval clot

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue–GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.

Monitoring and imaging the clot during systemic thrombolysis in stroke patients

Intravenous thrombolysis is the only approved treatment for acute ischemic stroke when administered within the first 3 h of stroke onset. Response to systemic thrombolysis depends on several factors including the location of arterial occlusion, clot characteristics and, ultimately, the embolic source. In the last few years, tremendous progress has been made, resulting in the widespread implementation of noninvasive neurovascular techniques. These imaging modalities are being increasingly performed in the acute stroke setting, without substantial delay, in a large number of centers worldwide. Transcranial Doppler ultrasound provides a unique opportunity to assess several aspects of clot dissolution by means of continuous monitoring of recanalization during and after tissue plasminogen activator administration. This approach allows for the evaluationof patients at the bedside and in real time due to the commencement, timing, speed and degree of artery reopening in addition to allowing the documentation of reocclusion after successful recanalization. Gradient refocused echo susceptibility vessel sign (GRE SVS) magnetic resonance imaging may be particularly useful for the identification of an intravascular thrombus during the acute phase of ischemic stroke; GRE SVS may represent a surrogate of clot composition and differential response to thrombolysis. The increasing availability of advanced neurovascular techniques may, in the near future, improve the design of stroke trials. The capability of these techniques to assess not only tissue viability but also key aspects regarding susceptibility to thrombolysis such as location, amount, composition, and age of the offending clot may improve the safety and efficacy profile of thrombolytic therapy for acute ischemic stroke.

Profibrinolytic activity of the direct thrombin inhibitor melagatran and unfractionated heparin in platelet-poor and platelet-rich clots

Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.

Significance of Susceptibility Vessel Sign on T2*-Weighted Gradient Echo Imaging for Identification of Stroke Subtypes

In contrast to platelet-rich white thrombi, red thrombi in the heart are rich in fibrin and trapped erythrocytes. The magnetic susceptibility effect of deoxygenated hemoglobin in red thrombi may result in hypointense signals on T2*-weighted gradient echo imaging (GRE). We tested the hypothesis that a GRE susceptibility vessel sign (SVS) is specific for cardioembolic stroke. This retrospective study examined data from acute ischemic stroke patients who underwent diffusion-weighted imaging, GRE and magnetic resonance angiography (MRA) within 24 hours of stroke onset and who had symptomatic occlusion of large intracranial arteries in the circle of Willis. Hypointense signals within vascular cisterns on GRE corresponding to symptomatic vascular occlusion were termed "GRE SVS." Recanalization was assessed on follow-up MRA performed within 7 days of onset. The relationships between GRE SVS and stroke subtypes and subsequent recanalization were explored. Of the 95 patients who met the inclusion criteria, GRE SVS was observed in 45 (47.4%). GRE SVS was more commonly associated with cardioembolic stroke patients (31 of 40, 77.5%) than with other stroke subtypes (14 of 55, 25.5%; P<0.001). In 66 patients who underwent follow-up MRA, GRE SVS was associated with subsequent recanalization (P<0.001). Multivariate analysis showed that GRE SVS was an independent predictor of cardioembolic stroke and subsequent recanalization (odds ratio, 10.75 and 4.26; 95% CI, 3.68 to 31.47 and 1.12 to 16.30). GRE SVS may predict cardioembolic stroke and subsequent recanalization. Identifying clot composition may be important in choosing the optimal treatment based on clot characteristics.

Biochemical characterization of autologous fibrin sealants produced by CryoSeal ® and Vivostat ® in comparison to the homologous fibrin sealant product Tissucol/Tisseel ®

Different principles for production of “autologous fibrin sealant” have been established, and commercial devices employing these methods are nowadays available and used in clinical routine. Users might anticipate for these autologous fibrin sealants features comparable to commercial homologous fibrin sealants, used in surgical routine for many years. However, only little is known about biochemical properties, formation, cross-linking and stability of fibrin sealant clots produced for autologous use with the aid of commercially available devices. We have investigated protein composition, formation and stability of clots obtained from autologuous fibrin sealants produced with commercially available devices (CryoSeal ® and Vivostat ®) and compared these parameters to those of the industrially produced homologous fibrin sealant Tissucol/Tisseel ®. The CryoSeal ® product is a mixture of many plasma proteins; the Vivostat ® product and Tissucol/Tisseel ® appear as comparatively pure plasma derivatives. The products differ in their protein composition and concentrations, including their concentration in fibrin. Significant fibrin α and γ -chain cross-linking by FXIIIa occurs only in Tissucol/Tisseel ® clots. In test tubes CryoSeal ® and Vivostat ® (tranexamic acid-free formulation) fibrin clots liquefy within 1–2 days, but Vivostat ® (tranexamic acid containing formulation) clots were stable for 4 days and showed partial liquefaction after 5 days. Tissucol/Tisseel ® clots, containing the protease inhibitor aprotinin, appeared unchanged over the observation period of 5 days. In an in vitro model mimicking in vivo conditions (diffusion of protease inhibitors and proteolytic digestion) clot liquefaction occurs at day 1 for all autologous fibrin sealants clots, with an observable delay for the tranexamic acid containing Vivostat ®, and day 5 for Tissucol/Tisseel ® clots. Characterization of the CryoSeal ® and Vivostat ® fibrin sealants and Tissucol/Tisseel ® and their performance show a clear difference in biochemical properties.

Approval of the MERCI Clot Retriever

Section Editors: Marc Fisher MD Antoni Davalos MD The Food and Drug Administration (FDA) evaluates applications for new human drugs, biologics, and complex medical devices. Companies must obtain FDA approval to legally market these products. In August, the FDA gave Concentric Medical clearance to market its Merci Retriever system to “remove blood clots from the brain in patients experiencing an ischemic stroke.” Given that the FDA is charged with “protecting the public health by assuring the safety, efficacy, and security of… biological products and medical devices…, ” “advancing public health by helping to speed innovations that make medicines … more effective, safer, and more affordable,” and “helping the public get the accurate, science-based information they need to use medicines … to improve their health,”1 the FDA’s decision to approve the Merci Retriever system is of concern. The pathways to approval are reviewed by Felten et al in the accompanying article and are outlined in Figure 1. Figure 1. Potential pathways for device approval. The decision to approve the Merci Retriever was based on data from the MERCI (Mechanical Embolus Removal in Cerebral Ischemia) Trial; the approval was granted through the 510(k) process. The Merci Retriever system includes a flexible nickel titanium (nitinol) wire that obtains a helical shape once it is passed through the tip of the guidance catheter. In practice, the catheter/wire is passed distal to the thrombus, the catheter is removed, and the helical configuration assumed by the wire; the clot is then trapped in the helix and withdrawn from the vasculature (Figure 2). The 510(k) clearance means that the Merci Retriever was felt to be substantially equivalent to a predicate device. In this case, the predicate device was the Concentric Retriever, which itself received 510(k) clearance by the FDA in May 2001 for “use in …

No Effect of Clot Age or Thrombolysis on Argatroban's Inhibition of Thrombin

The purpose of this study was to establish the effects of clot age and thrombolysis, with either streptokinase or tissue-type plasminogen activator (tPA), on argatroban's ability to inhibit thrombin. The antithrombotic activity of argatroban has been quantified in fibrin clot permeation and fibrin clot perfusion systems as a function of clot age and composition. Analysis of the argatroban dose-response data with a competitive inhibition model has yielded IC50 values in the low micromolar range. Results obtained in a plasma clot permeation system have also shown that argatroban is a potent inhibitor of clot-bound thrombin, independent of either clot age or the presence of hemostatically active platelets. Treatment of aged plasma clots with either streptokinase or alteplase, at therapeutic levels, increased the available thrombin activity, yet argatroban still inhibited this clot-associated thrombin with IC50 values in the low micromolar range. Scanning electron microscopy/morphometric analyses demonstrated that permeation with argatroban had no significant effects on clot structure. We conclude that argatroban is an effective inhibitor of thrombin bound to aged fibrin clots, in purified systems and in plasma clots, as well as in clots that have been treated with the thrombolytic agents streptokinase and alteplase.© 1998 by The American Society of Hematology.

No Effect of Clot Age or Thrombolysis on Argatroban’s Inhibition of Thrombin

The purpose of this study was to establish the effects of clot age and thrombolysis, with either streptokinase or tissue-type plasminogen activator (tPA), on argatroban’s ability to inhibit thrombin. The antithrombotic activity of argatroban has been quantified in fibrin clot permeation and fibrin clot perfusion systems as a function of clot age and composition. Analysis of the argatroban dose-response data with a competitive inhibition model has yielded IC50 values in the low micromolar range. Results obtained in a plasma clot permeation system have also shown that argatroban is a potent inhibitor of clot-bound thrombin, independent of either clot age or the presence of hemostatically active platelets. Treatment of aged plasma clots with either streptokinase or alteplase, at therapeutic levels, increased the available thrombin activity, yet argatroban still inhibited this clot-associated thrombin with IC50 values in the low micromolar range. Scanning electron microscopy/morphometric analyses demonstrated that permeation with argatroban had no significant effects on clot structure. We conclude that argatroban is an effective inhibitor of thrombin bound to aged fibrin clots, in purified systems and in plasma clots, as well as in clots that have been treated with the thrombolytic agents streptokinase and alteplase.© 1998 by The American Society of Hematology.

418 Fibrinolysis by urokinase: Effect of clot composition on the lysis kinetics

418 Fibrinolysis by urokinase: Effect of clot composition on the lysis kinetics

Study of thrombus from thrombosed hemodialysis access grafts.

To determine the volume and composition of clot within thrombosed hemodialysis access grafts. Clots were collected in 22 patients at surgical thrombectomy of polytetrafluoroethylene grafts. Histologic analysis was performed in 10 of these clots plus 21 randomly selected clots from the pathology archives. A small, firm piece of whitish thrombus ("arterial plug") was almost always recovered from the arterial limb of the graft. This plug had a concave surface and ranged from 5 mm to 3 cm in length. The remaining clot was soft, red thrombus. The mean weight of all clots was 3.4 g, and mean volume was 3.2 cm3. Average graft length was 42 cm. Histologically, the arterial plug had a characteristic appearance of densely compacted alternating layers of erythrocytes and fibrin. Clot volume in thrombosed dialysis grafts is much less (approximately equal to 25%) than would be expected if the graft were completely filled with thrombus, a finding of significance to mechanical thrombolytic techniques. Resistance of the arterial plug to pulse-spray thrombolysis is likely due to compaction.

Thrombolytic therapy in acute ischemic stroke

Data from two large multi-center studies show that thrombolysis can improve outcomes in some patients with acute ischemic stroke. Hemorrhage remains a concern, as does finding ways to increase the proportion of patients who respond to treatment. To be effective, clot lysis must be accompanied by re-establishing local perfusion of viable brain tissue. New technical developments will facilitate the application of intra-arterial thrombolysis and angioplasty for these purposes. More research is needed into the factors which predict good or bad responses to thrombolysis and into clot composition and hemodynamic flow dynamics.

Comparative evaluation of clinically available inferior vena cava filters with an in vitro physiologic simulation of the vena cava.

A physiologic in vitro model of the human inferior vena cava (IVC) was developed to evaluate the clot-trapping efficiency of various IVC filters. The flow model closely simulates the physical parameters of the human IVC in fluid viscosity, specific gravity, temperature, and pulsatility; blood flow velocity, volume, and inherent turbulence; and vein orientation, wall compliance, and clot composition. Five filters--Greenfield (G), Bird's Nest (BN), Vena Tech (VT), Simon nitinol (SN), and titanium Greenfield (TG)--were compared by using two vena cava and blood clot sizes and horizontal and vertical orientation of the model. Each filter varied significantly in its clot-capturing efficiency, depending on vena cava size and orientation and emboli size. Overall, decreasing rank order in filter clot-trapping efficiency was SN, BN, VT, TG, and G. The in vitro model of the human IVC is an alternative to multicenter clinical trials in evaluating the relative clot-trapping efficiency of various IVC filters.

Composition of emboli influences the efficacy of thrombolysis with rt-PA in a rat stroke model

Background and purpose: The efficacy of thrombolysis with recombinant tissue plasminogen activator and its dependency upon clot composition was tested in an embolic stroke model. Methods: In 53 rats the carotid territory was embolised with two arterial-like type of clots and one venous-like type of clot. The arterial ones were either fibrin poor (group A) or fibrin rich (group B). Hemispheric cerebral blood flow before and after embolisation was measured by intraarterial 133 Xenon injection method. 15 min after embolisation 18 animals were treated with tissue plasminogen activator 20mg/kg, and 35 animals with saline. Carotid angiography displayed the rate of occlusion of the cerebral arterial supply before and after treatment. Brains were fixed after 2 days, evaluated neuropathologically and infarct volume measured. Results: Cerebral blood flow was reduced by 36 to 62% after embolisation. The comparison of post-treatment angiography of treated animals to controls in group embolised with arterial-like fibrin rich clots showed significant (p=0.0163) reperfusion in thrombolytic treated animals. Thrombolytic therapy significantly reduced the infarct volume from 11.6 to 1.9% of embolised hemisphere volume (p=0.019) in group embolised with fibrin-poor clots and from 18.3 to 0.0% (p=0.0001) in the group embolised with fibrin-rich clots. Among the treated animals embolised with fibrin rich clots, both the completely recanalised animals (p=0.0003) and the partially recanalised animals (p=0.027) developed smaller infarctions than control animals. No hemorrhagic complications were observed. The control animals embolised with venous-like clots recanalised spontaneously and developed almost no infarctions and only temporary clinical damage. Conclusions: Early thrombolytic therapy induced recanalisation and reduced infarct volume after embolic stroke, this effect was particularly apparent in the group embolised with fibrin-rich clots.

Magnetic Resonance Imaging of Blood and Clots In Vitro

The effects of variations in blood clot composition on magnetic relaxation rates and magnetic resonance (MR) image have been characterized in vitro. Both 1/T1 and 1/T2 were found to be linear functions of hematocrit for blood and clot, with increases in hematocrit resulting in progressive decreases in image signal intensity. Clot formation in fully oxygenated samples produced no change in relaxation rates or MR images compared with unclotted blood, but clot retraction was associated with a significant increase in 1/T2 that resulted in a decreased signal. Retraction resulted in a heterogenous image with appearance of a hypointense peripheral rim; differences in the method of clot preparation resulted in significant image inhomogeneity. The pattern of fibrinolysis was found to depend on the type of plasminogen activator used and its site of initial application. Injection of tissue plasminogen activator into the clot resulted in lysis, primarily in the clot interior, whereas placing the enzyme in the surrounding serum caused degradation from the outside of the clot. Both observations are consistent with the high binding affinity of tissue plasminogen activator for fibrin. By comparison, streptokinase, with low fibrin binding affinity, dissolved thrombi in a peripheral pattern whether injected into the thrombus or introduced in the serum. These findings identify several variables of clot composition and structure that influence MR images of thrombi and should be considered in their interpretation.

Protamine-induced platelet aggregation and clotting investigated by ultrasound.

Using high-resolution real-time ultrasound to monitor platelet aggregation and plasma clotting, the effect of protamine on platelets was evaluated in a dynamic system of plasma in vitro. Protamine induced platelet aggregation preceding plasma clotting at both low (1.6 s-1) and moderate (22.6 s-1) shear rates. The onset of aggregation and clotting was accelerated at the higher shear rate. Low-shear clots were heterogeneous on ultrasonic imaging, whereas moderate-shear clots were more densely homogeneous. Protamine-induced platelet aggregation was reversed by additional heparin. Pretreatment of plasma with prostacyclin prevented protamine-induced aggregation, but clot formation occurred. However, such clots were less echogenic. EGTA and hirudin inhibited both aggregation and clotting. Our ultrasonic aggregometry showed that platelet aggregation was induced by protamine before clots formed, and that not only shear rate but platelet activation might affect the rate and composition of plasma clots.

Effects of Dextran on the Molecular Structure and Tensile Behaviour of Human Fibrin

Characteristic changes induced by dextran during the conversion of fibrinogen to fibrin have previously been shown to be associated with profound alterations in morphology of fibrin. However, whether dextran is incorporated into the fibrin molecule and whether morphological changes are associated with alterations in mechanical behaviour of formed fibrin was unclear. The investigations described show that the fibrin made in the presence of dextran has a shortened syneresis time, a lowered modulus of elasticity, an increased elongation and diminished ultimate strength at break. The molecular composition of fibrin clots remains unaltered despite the altered mechanical properties and morphological changes. Furthermore, dextran is not incorporated into the fibrin structure in any appreciable quantity. It is suggested that these several effects of dextran on clot morphology, tensile behaviour and kinetics of fibrin formation arise from increased forces of attraction between fibrin molecules such that fibrin chains are held together by weak secondary cross-links rather than by stronger primary cross-links which are hidden within the thicker fibrin chain bundles.

Factors affecting the viscosity of hyaluronic acid and synovial fluid

The composition of clots formed from bovine synovial fluids was found to vary markedly with pH at 0.05 ionic strength. All hyaluronic acid was found in the clot over the range pH 2.2–4. The percentage of hyaluronic acid in the clot increased with a decrease of pH. At ionic strengths greater than about 0.22, mucin clot formation did not occur. These mucin clots belong to the class of complex coacervates of Bungenberg de Jong and appear to be combinations of a negatively charged acid (hyaluronic acid) and positively charged proteins. Synovial fluids underwent a mainly irreversible loss of viscosity upon exposure to acid conditions, especially in the pH interval 3–4. This loss in viscosity appears to have arisen from the oxidative-reductive depolymerization (ORD) reaction and a depolymerization of hyaluronic acid. The effect of pH on the ORD reaction was studied and found to be different for the four “reducing agents” studied. Three of four “reducing agents” showed a maximum effect in the range pH 4–7.

The mucin of bovine sublingual glands

Extracts of bovine sublingual glands were fractionated and the materials (mucin) responsible for the viscous stringy properties of the extracts were extensively purified. The progress of purification was followed by electrophoretic, ultracentrifugal and viscometric measurements. The purest product contained 23.3 per cent N-acetyl-hexosamine, 17.8 per cent sialic acid, 7.6 per cent 6-deoxyhexose, 20.6 per cent hexose (as galactose), and about 25 par cent protein. The molar ratios were approximately: hexosamine, 2; sialic acid, 1; 6-deoxyhexose, 1; hexose as galactose, 2. The electrophoretic mobility of the product was −4.6 × 10 −5cm 2/sec/V at pH 7.4, ionic strength 0.1. The sedimentation constant was 8.6 S in the same buffer. The composition of the mucin clot was also investigated and found to be a dissociable mucoprotein. The mucin appears to act as one of the anionic components in the formation of the mucin clot. The purest mucin itself gave no clot by adjustment of the pH to 3.5 except in the presence of added serum albumin.

The composition and properties of the mucin clot from cattle submaxillary glands

Mucin clots from cattle submaxillary glands were lyophilized as sodium salts with retention of the viscosity of the extracts and of solubility in water. The viscosity was 25.8 cP in 1% aqueous solution and 4.5 cP in 1% phosphate buffer of pH 7.4 and ionic strength 0.1. The specific rotation was [ α] D 25 = −23.2 ° (0.87 per cent in water). The mucin contained about 13 per cent sialic acid, 11 per cent hexosamine (8 per cent galactosamine and 3 per cent glucosamine as the acetyl derivatives) and 75 per cent protein. The molar ratio of galactosamine to sialic acid was unity. The mucin showed four peaks when subjected to electrophoresis at pH 10 in a phosphate buffer of ionic strength 0.1. The average mobilities were −7.2, −8.0, −10.3 and −11.3 × 10 −5 cm 2/sec per V, respectively, at pH 10. Three components were evident at pH 7.4 and 8.0, and only two at pH 6. The major component had a mobility of −6.3 × 10 −5 at pH 7.4. Two ultracentrifugal components of 2.1 and 2.5 S were evident at pH 7.4. At pH 10, a small more rapidly moving 5.0 S component was also present.