Increased platelet, but unaltered fibrinogen, accumulation in experimental thrombi in alloxan-induced diabetic rabbits.

Platelets from diabetic humans and animals have been found previously to be hypersensitive to agonists, including thrombin, in vitro but it is unclear if this hypersensitivity also occurs in vivo and leads to a greater thrombotic tendency. In the present study, the effect of diabetes was examined on thrombus formation and vessel wall responses which result from continuous intimal injury induced by indwelling aortic catheters in rabbits. Platelet and fibrin(ogen) associated with the thrombus and damaged aortae were examined. Control or alloxan-induced diabetic rabbits (9-12 months after initial treatment) were injected with 51Cr-labeled autologous platelets and 125I-labeled fibrinogen (prepared from control rabbits) before insertion of indwelling aortic catheters. The anesthetized rabbits were perfused-fixed after 20 hr or 4 days. The dry weight of thrombus that formed was determined and platelet and fibrin(ogen) accumulation in thrombi and on injured aortae were calculated from the associated 51Cr and 125I, respectively. In diabetic rabbits, more platelets accumulated in the thrombi which formed after either 20 hr or 4 days, although the weight of thrombus and net fibrin(ogen) incorporation into the thrombus were not different from corresponding control rabbits. Net platelet and fibrin(ogen) association with the injured aortae were not different between control and diabetic rabbits. It is likely that the increased platelet accumulation in arterial thrombi in diabetic rabbits which results from continuous injury to aortae is a consequence of hypersensitivity of these platelets to thrombin generated in the thrombus and at the sites of vessel injury.     

Real-time analysis of platelet aggregation and procoagulant activity during thrombus formation in vivo

The exact mechanism of blood vessel thrombus formation remains to be defined. Here, we introduce a new approach to probe thrombus formation in blood vessels of living animals using intravital microscopy in green fluorescent protein (GFP)-transgenic mice to simultaneously monitor platelet aggregation and procoagulant activity. To this end, GFP-expressing platelets and annexin A5 labeled with a fluorescent dye were employed to visualize and analyze platelet aggregation and markers of procoagulant activity (platelet surface phosphatidylserine (PS)). Laser-induced thrombi increased and then decreased in size with time in vessels of living animals, whereas platelet surface PS initiated at the site of injury and then penetrated into the thrombus. PS-positive platelets were predominantly localized in the center of the thrombus, as was fibrin generation. The experimental system proposed here is a valuable tool not only for investigating mechanisms of thrombus formation but also to assess the efficacy of antithrombotic drugs within the vasculature.     

Pathophysiology of atherothrombosis: Mechanisms of thrombus formation on disrupted atherosclerotic plaques

Atherothrombosis is a leading cause of cardiovascular mortality and morbidity worldwide. The underlying mechanisms of atherothrombosis comprise plaque disruption and subsequent thrombus formation. Arterial thrombi are thought to mainly comprise aggregated platelets as a result of high blood velocity. However, thrombi that develop on disrupted plaques comprise not only aggregated platelets, but also large amounts of fibrin, because plaques contain large amount of tissue factor that activate the coagulation cascade. Since not all thrombi grow large enough to occlude the vascular lumen, the propagation of thrombi is also critical in the onset of adverse vascular events. Various factors such as vascular wall thrombogenicity, local hemorheology, systemic thrombogenicity and fibrinolytic activity modulate thrombus formation and propagation. Although the activation mechanisms of platelets and the coagulation cascade have been intensively investigated, the underlying mechanisms of occlusive thrombus formation on disrupted plaques remain obscure. Pathological findings derived from humans and animal models of human atherothrombosis have uncovered pathophysiological processes during thrombus formation and propagation after plaque disruption, and novel factors have been identified that modulate the activation of platelets and the coagulation cascade. These findings have also provided insights into the development of novel drugs for atherothrombosis.     

Acute administration of ethanol to rabbits inhibits thrombus formation induced by indwelling aortic catheters

Platelet aggregation and secretion of granule contents in response to specific agonists are inhibited by acute ethanol (1 to 4 mg/ml in vitro or 4 to 5 mg/ml ex vivo). However, acute administration of ethanol, giving blood levels of 4 to 5 mg/ml, does not affect platelet adherence to de-endothelialized rabbit aortae in vivo, which is the initial step in thrombus formation. Whether ethanol inhibits subsequent reactions in thrombus formation induced by indwelling catheters in rabbit aortae was investigated. Before insertion of the catheters, the rabbits received injections of 51Cr-labeled platelets (and, in some experiments, [125I]fibrinogen), and were given ethanol by stomach tube, to achieve blood levels of approximately 4 mg/ml. Thrombus formation after 3 hours was assessed by determining the number of platelets and the amount of radiolabeled fibrin(ogen) associated with the aortae, and by determining the thrombus weights; all three measurements indicated a 64 to 84% reduction in thrombosis. Thus, experimentally induced thrombus formation in the aorta is significantly reduced by ethanol.     

Platelets, inflammatory cells, von Willebrand factor, syndecan-1, fibrin, fibronectin, and bacteria co-localize in the liver thrombi of Bacillus anthracis-infected mice

Vascular dysfunction and thrombosis have been described in association with anthrax infection in humans and animals but the mechanisms of these dysfunctions, as well as the components involved in thrombi formation are poorly understood. Immunofluorescent microscopy was used to define the composition of thrombi in the liver of mice challenged with the Bacillus anthracis Sterne spores. Lethal infection with the toxigenic Sterne strain, in contrast to the non-lethal, non-toxigenic delta-Sterne strain, demonstrated time-dependent increase in the number of vegetative bacteria inside the liver sinusoids and central vein. Massive appearance of thrombi typically occluding the lumen of the vessels coincided with the sudden death of infected animals. Bacterial chains in the thrombi were stained positive for syndecan-1 (SDC-1), fibronectin, and were surrounded by fibrin polymers, GPIIb-positive platelets, von Willebrand Factor (vWF), CD45-positive leukocytes, and massive amount of shed SDC-1. Experiments with human umbilical vein endothelial cells (HUVECs) demonstrated the active role of the host response to the secreted pathogenic factors of bacteria during the onset of the pro-thrombotic condition. The bacterial culture supernatants, as well as the isolated proteins (the pore-forming toxin anthrolysin O and phospholipase C) induced release of vWF, while anthrolysin O, sphingomyelinase and edema toxin induced release of thrombin from HUVECs and polymerization of fibrin in the presence of human plasma. Conclusion: Our findings suggest that activation of endothelium in response to infection can contribute to the formation of occlusive thrombi consisting of aggregated bacteria, vWF, shed SDC-1, fibrin, activated platelets, fibronectin and leukocytes.     

Thrombogenesis of the Rabbit Arterial Plaque: An Electron Microscopic Study

Rabbit arteries, de-endothelialized with an intravascular balloon catheter and allowed to heal for 4 weeks, showed intimal changes that were similar to the preatherosclerotic fibromusculoelastic plaques of man. Reinjury of the healed vessels by balloon catheter produced marked quantitative and qualitative alterations of hemostasis, as compared to that in previously uninjured vessels. The most apparent modification of thrombogenesis 10 minutes after injury to the plaque was a large increase in the size of the thrombotic deposits. Features of this exaggerated response were the major participation of fibrin in thrombus formation and greater platelet accumulation. Some platelets and fibrin strands appeared to penetrate into and beneath the neointima. By 3 hours, these deposits had diminished in size, although the hemostatic mass remained larger in the doubly injured vessels.     

The structure and growth of valve-pocket thrombi in femoral veins

The structure of 50 small thrombi in femoral valve pockets and the microscopic contents of 35 apparently empty pockets were studied in an attempt to ascertain the nature of the microscopic nidi from which thrombi form and their manner of growth to visible thrombi. Sixteen thrombi had little or no cellular invasion. Most of these recent structures had two main regions, red areas restricted distally in the pocket by the vein wall, and larger white regions comprising most of the thrombus length and often covering the red areas. Red areas are the early sites of cellular adhesion and invasion and the likely sites of origin of most thrombi. They were usually dominated by red cells and fibrin. White zones, which represent propagation growth, are characterized by many foci of platelets with fibrin borders (platelet-fibrin units). Some red areas also contained platelet-fibrin units but they were few and tiny; platelets were not seen in others and one small wholly red thrombus was devoid of platelets. Degenerative changes in platelet-fibrin units were observed, and it is postulated that many become purely fibrin structures. There was no significant evidence of preceding intimal damage in the vein wall. Therefore nidi are laid down on normal endothelium probably on the vein wall near the apex of the pocket. Some pockets, empty of thrombi, contained condensed foci of red cells or tiny fibrin fragments surfaced by endothelial cells and considered to be the remnants of aborted thrombi; a few contained clumps of platelets or leucocytes. It is postulated that any of these may represent the nidi from which thrombi grow. Several thrombi also incorporated large fat droplets, numerous in two. Fat embolic globules derived from fractures are their likely source.     

Thrombosis induced in vivo in the mesenteric artery of normal and thrombocytopenic rats, an electron-microscopic study of the early arterial wall reaction

Arteries taken from 1 to 3 days after local thrombosis had been induced by the passage of a weak electric current followed by the topical administration of ADP were studied by electronmicroscopy. The characteristic changes observed during this period included total disappearance of pre-existing thrombi, rapid reconstitution of the endothelium and important hypertrophy of smooth muscle cells in the media. Thrombocytopenia induced by the administration of antiplatelet serum produced no alterations in untreated arteries. When it was coupled to thrombus induction, healing of injured endothelial and smooth muscle cells was grossly impaired, hypertrophy of the media was absent and fibrin infiltrated the arterial wall at the site of thrombus induction. These results bring support to the view that blood platelets play an important role in the induction of the regeneration response of injured arterial cells.     

Thrombosis induced in vivo in the mesenteric artery of normal and thrombocytopenic rats,an electron-microscopic study of the early arterial wall reaction.

Arteries taken from 1 to 3 days after local thrombosis had been induced by the passage of a weak electric current followed by the topical administration of ADP were studied by electronmicroscopy. The characteristic changes observed during this period included total disappearance of pre-existing thrombi, rapid reconstitution of the endothelium and important hypertrophy of smooth muscle cells in the media. Thrombocytopenia induced by the administration of antiplatelet serum produced no alterations in untreated arteries. When it was coupled to thrombus induction, healing of injured endothelial and smooth muscle cells was grossly impaired, hypertrophy of the media was absent and fibrin infiltrated the arterial wall at the site of thrombus induction. These results bring support to the view that blood platelets play an important role in the induction of the regeneration response of injured arterial cells.     

Localization of Ferritin-Conjugated Anti-Fibrin/Fibrinogen in Platelet Aggregates Produced In Vitro

A ferritin-conjugated anti-fibrin/fibrinogen was localized by means of light and electron microscopy in artificial in vitro thrombi formed in the presence of the labeled antibody, and in preformed ADP-induced platelet aggregates. The ferritin was distributed throughout the central and peripheral regions of the columns of aggregated platelets in the thrombi. In the preformed ADP aggregates, ferritin was deposited only by infiltration from surrounding plasma and was confined to the periphery of the columns. The even distribution of ferritin in the central zone of the platelet columns of the thrombi indicated a specific reaction had occurred before or during thrombus formation unrelated to infiltration of plasma. In the artificial thrombi, the ferritin-labeled antibody was localized on the surface layer of platelets and on the bridging structures composed of the combined surface layers in the narrow spaces between cohesive platelets. Vesicles and alpha granules within the platelets also were tagged. The absence of obvious fibrin between narrow interspaces and within the platelets indicated that the antibody had reacted with fibrinogen or partly polymerized fibrin at these sites. Many invaginations of the platelet membrane containing dense fibrillar material were interpreted to be alpha granules discharging their contents during the “release reaction” at the time of aggregation. This material, which was tagged by the ferritin-conjugated antibody, merged with the interplatelet bridges to suggest that released fibrinogen from within the platelet contributed to the structural bond and strengthened it. A layer of dense fibrin and altered platelets in the periphery of the columns of aggregated platelets in the artificial thrombi contained the platelets and limited further growth of the aggregates. The fibrin was thought to be derived from infiltrated plasma as well as from released intraplatelet fibrinogen. Platelet fibrinogen thus appeared to take part both in the cohesion of aggregated platelets and in the stabilization of the aggregates formed.     

Immunohistochemical study on the liver in autopsy cases with disseminated intravascular coagulation (DIC) with reference to clinicopathological analysis

Summary Immunohistochemical and clinicopathological studies were performed in 27 autopsy cases with indisputable DIC, which had been selected from 1,800 autopsy cases of elderly people based on the following two criteria; 1. presence of fibrin thrombi in glomeruli, and 2. presence of fresh patchy necrotic foci in myocardium and/or fibrin thrombi in splenic sinuses. A high incidence of liver lesions (22/27) was revealed in autopsy cases with indisputable DIC. The liver lesions could be classified into four groups. Group-I (Central degeneration) was characterized by massive precipitation of fibrin irregularly around the central vein, causing parenchymal damage. Group-II (Central necrosis), showed coagulation necrosis in the cental zone due to circulatory disturbance caused by either shock as a cause of DIC or abrupt cessation of blood flow into the lobules following fibrin thrombus formation in vessels of Glisson's sheath. Both group-I and -II showed a short clinical duration of DIC. Group-III (Sinusoidal thrombosis), showed the presence of fibrin thrombi in sinusoids with mild parenchymal damage and long clinical duration of DIC. Group-IV (No thrombosis), showed neither parenchymal damage nor fibrin thrombi in sinusoids, but a long clinical duration of DIC.     

Arrest and extravasation of B16 amelanotic melanoma in murine lungs. A light and electron microscopic study

The arrest and extravasation of tail vein-injected B16 amelanotic melanoma (B16a) cells, disaggregated from subcutaneous tumors, were studied at intervals from 10 minutes to 5 days in lungs of C57BL6J mice. Tumor cells were found in the pulmonary vasculature at 10 minutes postinjection and were commonly associated with platelets and fibrin. Tumor cells with associated thrombi increased, reaching a peak at 4 hours. Arrest of the B16a melanoma tumor cells appears to involve contact with endothelial plasma membrane, often with adjacent but not interposed platelet and fibrin thrombus formation. The tumor cell-associated thrombi subsequently decreased in frequency and were rarely found after 48 hours. The arrested tumor cells were initially in contact with the endothelial cells, which were gradually displaced by tumor cells achieving contact with the vascular basal lamina (BL). Initial contact with the vascular BL was observed at 4 hours, with a progressive increase in contact over the subsequent 2 days. Blood flow was commonly reestablished past the BL-attached tumor cells after dissolution of the thrombi. Mitotic figures in the tumor cells attached to the BL were frequent after 24 hours and the tumor appeared to proliferate intravascularly along the basal lamina. Penetration of the BL by tumor cell cytoplasmic processes was first observed at 3 days with continued dissolution of the vascular BL developing through day 5. Extravasation occurred through a combination of intravascular tumor cell proliferation and destruction of vascular BL by the B16a cells. Migration or diapedesis of the tumor cells was not observed in any of the time periods studied.     

Naloxone modifies sugar-water intake in rats drinking with open gastric fistulas

The effects of the opiate antagonist naloxone (NX) on fluid preference and intake were determined in rats drinking with chronically indwelling gastric fistulas. The subjects were tested both after 22.5 hr fluid deprivation, and no deprivation, with open fistulas (sham drinking), as well as with closed fistulas. Following an injection of either saline or NX (0.5–10.0 mg/kg, administered SC), or no injection, the subjects were given the choice to drink water or 10% sucrose, in a two-bottle test, for 1 hr/day. With open fistulas, and following fluid deprivation, the animals sham drank both sucrose and water, but had a strong preference for sucrose. When not fluid deprived, the same animals sham drank sucrose almost exclusively. NX significantly reduced sucrose intake by the sham drinking animals, in both the deprived and not deprived conditions, but did not modify fluid preference. These data support the idea that NX modifies affective reactivity to palatable solutions, and that NX's antidipsogenic actions are not due to feedback from post-absorptional events.     

Localisation of fluorescence-labelled recombinant tissue-type plasminogen activator (rt-PA) on thrombi in vitro

Recombinant tissue-type plasminogen activator exhibits a high specific affinity for fibrin and is therefore of great value as a thrombolytic agent. However, its distribution after binding to a thrombus has not yet been demonstrated. In this study the binding of fluorescence-labelled rt-PA to human whole blood thrombi in vitro was investigated.After labelling with dichlorotriazinylaminofluorescein (DTAF) rt-PA retained fibrin affinity as well as enzymatic activity. Aged thrombi were incubated with increased concentrations of DTAF-rt-PA. Fluorescence in cryosections of the clots, as visualised by incidence microscopy, was observed only at the thrombus surface. The following control experiments confirmed that the fluorescence was specifically derived from the labelled protein: 1. after incubation of thrombi with DTAF alone no fluorescence could be observed in cryosections prepared from these thrombi; 2. incubation of a thrombus, exhibiting DTAF-rt-PA derived surface fluorescence, with unlabelled rt-PA resulted in the complete loss of surface fluorescence; 3. the fluorescence was randomly distributed throughout the whole cryosection if DTAF-rt-PA was present during thrombus formation.The findings suggest that during clot lysis, rt-PA binds initially to the thrombus surface but does not penetrate into the body of the clot. Thrombolysis is, therefore, likely to proceed via successive fibrin removal from the clot surface.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter.

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Platelet-wall interactions in continuum models of platelet thrombosis: formulation and numerical solution.

A model is developed to describe the formation of platelet thrombi in coronary-artery-sized blood vessels. It involves interactions among a viscous, incompressible fluid; populations of non-activated and activated platelets; activating chemicals; and the vessel walls. Adhesion of platelets to the injured wall and cohesion between activated platelets is modelled using distributions of elastic links which generate stresses that can influence the fluid motion. The first version of the model presented involves two spatial scales: the microscale of the platelets and the macroscale of the vessel. A closure approximation is introduced that allows essential microscale behaviour to be computed while eliminating the necessity to explicitly track events on this scale. Computational methods are presented that meet the diverse challenges posed by the coupled nonlinear partial differential equations of the model and by the complex geometry of the constricted vessels in which the thrombosis simulations are carried out. Simulation results demonstrate that the model can produce thrombi that grow to occlude the vessel, that shear-stress exerted by the fluid on the thrombi can modify their subsequent growth and cause remodelling of their shape through small-scale local changes or large-scale structural breakup.     

Platelet interaction with damaged rabbit aorta.

We have quantified the accumulation of 51Cr-labeled washed rabbit platelets on the subendothelium of rabbit aortae following injury with a balloon catheter. The amount of radioactivity that became associated with the damaged wall within 10 minutes of the injury did not change appreciably during the following 24 hours, indicating that there was little turnover of platelets on the injured vessel wall. In addition, by injecting 51Cr-labeled platelets into rabbits at different times after injury, it was possible to estimate the reactivity of the exposed surface to newly injected platelets. Scanning electron microscopy showed that a monolayer of platelets initially formed on the injured surface; the number of platelets associated with the surface decreased over the 7-day observation period. The amount of 51Cr associated with the injured vessel wall also diminished during this period. The ability of the damaged surface to attract fresh platelets gradually decreased during the 7 days following injury. Platelet survival in rabbits was not significantly reduced following the removal of the aortic endothelium (balloon catheter injury 66.3 +/- 12.2 hours, sham operated 72.1 +/- 7.2 hours, untreated controls 76.2 +/- 3.8 hours). Thus, in rabbits, it cannot be assumed that platelet survival provides an estimate of endothelial injury in all circumstances.     

COMPARATIVE IMMUNOPATHOLOGIC STUDIES OF THROMBOTIC THROMBOCYTOPENIC PURPURA, HEMOLYTIC-UREMIC SYNDROME AND DISSEMINATED INTRAVASCULAR COAGULATION

The vascular lesion of thrombotic thrombocytopenic purpura was characterized by two distinct types of changes in the arteriole of the heart and brain: (1) multiple incomplete vascular occlusions due to the subendothelial deposits composed of a homogeneous fibrin fibrinogen-like substance and platelets without the formation of polymerized fibrin; (2) vascular wall thickening due to the intramural granular deposits of IgM and β1C.
A patient with hemolytic-uremic syndrome 9 days after the onset of the disease, exhibited (1) the formation of fibrin-thrombi in the glomerular capillary lumens, and (2) the granular deposits of IgM and β1C along the glomerular capillary walls and in the mesangium. In contrast, the case in which the symptoms subsided five weeks after the onset exhibited neither fibrin-thrombus formation nor the deposits of IgM and βC in the glomeruli. The vascular lesion of disseminated intravascular coagulation accompanied by pancreas carcinoma was located mainly in the capillaries, and were characterized by the formation of numerous fibrin-thrombi. Although the glomeruli contained numerous thrombi, there was neitherendocapillary proliferation nor deposits of immunoglobulins and complement components.     

Platelet receptor signaling in thrombus formation

Platelet activation and subsequent thrombus formation at sites of vascular injury is crucial for normal hemostasis, but it can also cause myocardial infarction and stroke. The initial capture of flowing platelets to the injured vessel wall is mediated by the interaction of the glycoprotein (GP) Ib-V-IX complex with von Willebrand factor immobilized on the exposed subendothelial extracellular matrix. Tethered platelets are then able to bind to collagens through the immunoglobulin-like receptor GPVI and to initiate cellular activation, a process that is reinforced by G protein-coupled receptors stimulated by locally produced thrombin and soluble mediators released from activated platelets. These signaling events lead to a rise in the cytosolic Ca²⁺ concentration, rearrangement of the cytoskeleton, release of granule content, and functional upregulation of integrin adhesion receptors allowing firm adhesion and thrombus growth. Fully activated platelets also undergo a procoagulant conversion thereby facilitating coagulation and thrombus stabilization. This review summarizes the most important receptor systems and signaling mechanisms involved in platelet activation and thrombus formation with special focus on recent discoveries.