Role of Leukocytes in Blood Coagulation and the Generalized Shwartzman Reaction

IN spite of intensive investigation, many of the factors which initiate blood coagulation and thrombosis remain obscure. The generalized Shwartzman phenomenon, which is due at least in part to intravascular coagulation, is classically obtained by two appropriately spaced, sub-lethal, intravenous doses of endotoxin given to rabbits and is characterized by disseminated thrombi in lungs, kidney and spleen; the characteristic lesion in the kidneys is renal cortical necrosis. Although the Shwartzman phenomenon can be prevented by anticoagulation^{1, 2}, its mechanism remains obscure. Leukocytes have been implicated as the mediators but only indirect evidence is available¹. Leukocytes also possess procoagulant and anticoagulant activity^3–5, the former, however, has always been considered too weak to be physiologically significant or able to cause intensive intravascular clotting with defibrination. We now have evidence that endotoxin given to rabbits may endow their leukocytes with considerable procoagulant activity in vitro, sufficient to produce intravascular clots in various organs when infused to untreated normal rabbits.     

Thrombotic thrombocytopenic purpura: a syndrome of intravascular platelet consumption.

In four of five patients with thrombotic thrombocytopenic purpura (TTP) in whom serial tests of hemostatic function were performed, severe thrombocytopenia, normal plasma fibrinogen concentrations and mildly increased concentrations of fibrinogen/fibrin degradation products were observed. Widespread platelet thrombi were found in arterioles and capillaries. Fibrin could be seen around some of the platelet clumps and was the main component in a small number of the thrombi in two patients. The observations show that TTP is a disorder in which intravascular platelet consumption results in disseminated platelet thrombosis. The coagulation system is apparently activated secondarily to platelet aggregation and variable quantities of fibrin are incorporated into the thrombi. Clinical improvement resulted from combined therapy with corticosteroids, heparin and drugs that suppress platelet function.     

Association of Localized Hypersensitivity and In-Stent Neoatherosclerosis with the Very Late Drug-Eluting Stent Thrombosis

Background

Localized hypersensitivity reaction, delayed arterial healing, and neoatherosclerosis inside the stent have been suggested as the underlying pathologic mechanisms of very late stent thrombosis (VLST) of drug-eluting stent (DES). The present study sought to explore the prevalence of inflammatory cell infiltrates and evidence for fragments of atherosclerotic plaques in the aspirated thrombi in patients with DES VLST.

Methods and Results

From April 2004 to September 2012, 48 patients with stent thrombosis (ST) of DES underwent thrombus aspiration with retrieved material sufficient for the histopathologic analysis; early ST (EST, within 30 days): N = 17, late ST (LST, between 31 and 365 days): N = 7, and very late ST (VLST, >1 year): N = 24. Eosinophil fraction in the aspirated thrombi was significantly higher in patients with VLST (8.2±5.7%) as compared with those with EST (4.3±3.0%) and LST (5.5±3.8%) (P = 0.03). Eosinophil fraction in the aspirated thrombi was significantly higher in 12 VLST patients with angiographic peri-stent contrast staining (PSS) and/or incomplete stent apposition (ISA) by intravascular ultrasound than in 12 VLST patients without PSS or ISA (10.6±6.1% versus 5.8±4.1%, P = 0.03). Evidences for fragments of atherosclerotic plaques in the aspirated thrombi were observed only in 3 (13%) out of 24 patients with DES VLST.

Conclusions

Eosinophil fraction in the aspirated thrombi was significantly higher in patients with DES VLST as compared with those with EST and LST. Evidences for fragments of atherosclerotic plaques were relatively uncommon in patients with DES VLST.     

Diagnosis of deep vein thrombosis using autologous indium-III-labelled platelets.

Forty-eight patients who had undergone surgical reduction of a fractured neck of femur or in whom deep vein thrombosis was suspected clinically were studied by ascending phlebography and imaging after injection of autologous indium-111-labelled platelets to assess the accuracy and value of the radioisotopic technique in diagnosing deep vein thrombosis. Imaging was performed with a wide-field gammacamera linked with data display facilities. Phlebography showed thrombi in 26 out of 54 limbs examined and a thrombus in the inferior vena cava of one patient; imaging the labelled platelets showed the thrombi in 24 of the 26 limbs and the thrombus in the inferior vena cava. The accumulation of indium-111 at sites corresponding to those at which venous thrombi have been shown phlebographically indicates that this radioisotopic technique is a useful addition to methods already available for the detection of deep vein thrombosis.     

The nonspecific nature of fibrin thrombi in ischemic bowel disease.

Twenty cases of ischemic bowel disease were analysed to determine the frequency and significance of fibrin thrombi in this condition. Fibrin thrombi were present in all 10 patients with occlusive ischemic bowel disease and in 7 of the 10 patients with nonocclusive ischemic bowel disease. In addition, fibrin thrombi were noted in a wide variety of specific and nonspecific inflammatory bowel diseases and in acute appendicitis. We conclude that fibrin thrombi are a nonspecific feature of tissue necrosis and that their mere presence in the bowel should not be regarded as an expression of disseminated intravascular coagulation.     

Liver Transplantation in Man—IV,Haemorrhage and Thrombosis

Blood coagulation and fibrinolytic factors have been measured in 13 patients treated by liver transplantation. During operation intravascular coagulation and fibrinolysis were increased, but seldom to a degree which would cause abnormal bleeding. Measurement of the catabolism of radioactive fibrinogen showed that increased intravascular coagulation continued for long periods after the operation. Despite secondarily increased fibrinolysis, there was a high incidence of thrombosis. Treatment with anticoagulants or with fibrinolysis inhibitors may be valuable in these patients.     

Endocardial Endothelial Dysfunction Progressively Disrupts Initially Anti then Pro-Thrombotic Pathways in Heart Failure Mice

Objective

An experimental model of endocardial thrombosis has not been developed and endocardial endothelial dysfunction in heart failure (HF) is understudied. We sought to determine whether disruption of the endothelial anti-coagulant activated protein C (APC) pathway in CREB_A133 HF mice promotes endocardial thrombosis in the acute decompensated phase of the disease, and whether alterations in von Willebrand factor (vWF) secretion from HF endocardium reduces thrombus formation as HF stabilizes.

Approach and results

Echocardiography was used to follow HF development and to detect endocardial thrombi in CREB_A133 mice. Endocardial thrombi incidence was confirmed with immunohistochemistry and histology. In early and acute decompensated phases of HF, CREB_A133 mice had the highest incidence of endocardial thrombi and these mice also had a shorter tail-bleeding index consistent with a pro-thrombotic milieu. Both APC generation, and expression of receptors that promote APC function (thrombomodulin, endothelial protein C receptor, protein S), were suppressed in the endocardium of acute decompensated HF mice. However, in stable compensated HF mice, an attenuation occurred for vWF protein content and secretion from endocardial endothelial cells, vWF-dependent platelet agglutination (by ristocetin), and thrombin generation on the endocardial surface.

Conclusions

CREB_A133 mice develop HF and endocardial endothelial dysfunction. Attenuation of the anti-coagulant APC pathway promotes endocardial thrombosis in early and acute decompensated phases of HF. However, in stable compensated HF mice, disruptions in endothelial vWF expression and extrusion may actually reduce the incidence of endocardial thrombosis.     

New Radioisotope Test for Detection of Deep Venous Thrombosis in the Legs

A radiopharmsceutical product, labelled macroaggregates of albumin (M.A.A.), which is in use as a lung scintiscanning agent has been noted to have an affinity for venous thrombi. With this material and an inexpensive portable scintillation detector we have attempted to diagnose and localize thrombi in leg veins. The procedure is performed at the bedside and the result is available in 30 minutes. Thirty-one patients with clinical evidence suggestive of deep venous thrombosis in the legs were studied by the radioisotope method and by phlebography. There was agreement in 18 of 21 legs shown to contain thrombus on phlebography and in 9 of 10 legs shown to be free of thrombosis on phlebography. There was, however, lesser agreement on the site of thrombosis between the two methods. The ease of performing the test combined with the rapidity of obtaining results and accuracy in diagnosis suggests that the test has a clinical application.     

Clinical relevance of protein C

Protein C is, after activation by thrombin, a potent inhibitor of blood coagulation. An isolated deficiency of protein C increases the risk of thrombosis. The two forms of protein C deficiency, the heterozygous and the homozygous deficiency state, have different clinical features. Patients with heterozygous protein C deficiency are at a high risk to develop venous thrombosis and pulmonary embolism. In newborns with homozygous protein C deficiency with very low protein C levels (1%) a purpura fulminans like syndrome was observed. Heparin and coumarin derivatives are effective drugs in heterozygous protein C deficiency, homozygous patients may be treated either by replacement of protein C or coumarin derivatives. Decreased protein C levels were observed in various other diseases: Chronic and acute liver disease, disseminated intravascular coagulation, malignancy, postoperatively and during treatment with asparaginase. The role of protein C in these diseases to trigger thrombosis is not yet established.     

Heparin and phenprocoumon inhibit kappa-carrageenin induced thrombosis in rats

The tail thrombosis after kappa-carrageenin injection in rats was significantly inhibited by heparin and phenprocoumon. Heparin was yet effective at doses as low as 150 IU/kg. The present data point to an involvement of blood coagulation in this new model. Injection of cobra venom factor with complement depletion caused no thrombosis inhibition. The differences between the present kappa-carrageenin induced thrombosis model and a disseminated intravascular coagulation after lambda-carrageenin are discussed.     

Indium-111 labelled platelets in diagnosis of leg-vein thrombosis: preliminary findings.

Platelets from eight patients thought clinically to have deep venous thrombosis were labbelled with indium-111 and reinjected. Subsequent scanning of the patients with a wholebody scanner and imaging with a gammacamera showed focal accumulation of the label at five sites in four legs, which correlated precisely with the sites of venous thrombi identified by ascending venography. This technique is a useful addition to methods for diagnosis venous thrombosis.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

In vivo monitoring of thrombosis in mice by optical coherence tomography

The objective of this study is to establish a novel method for continuously monitoring thrombus progression with various outcome measures and to assess the efficacy of antithrombotic drugs in murine thrombosis model in mice. In the study, thrombus was induced in the femoral vein of mice by FeCl₃ and monitored over time by spectral‐domain optical coherence tomography (OCT). Three‐dimensional images of thrombi with or without heparin as an antithrombotic agent were obtained from OCT angiography. In addition, several parameters of thrombi were analyzed and compared between control and anticoagulant groups. By using OCT, we were able to trace thrombus generation in the same mouse in real time. We found that in our model heparin reduced thrombus size by ~60% and thrombus cross‐sectional area by 50%. OCT results also show that both time to thrombus size (>0.02mm³) and time to occlusion (>30%) were significantly reduced after heparin addition. This study demonstrates that OCT reliably monitors thrombus generation and progression from various aspects including thrombus size. This enables us to measure the kinetic of thrombosis more accurately, and effectively evaluate the efficacy and activities of antithrombotic drugs. This model may represent a useful tool in antithrombotic drug discoveries in preclinical studies.      

Detection of Tissue Factor Antigen and Coagulation Activity in Coronary Artery Thrombi Isolated from Patients with ST-Segment Elevation Acute Myocardial Infarction

Introduction

Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.

Methods

Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.

Results

Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.

Conclusions

Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.     

Intracardiac thrombosis and aortic dissecting aneurysms in mustached tamarins (Saguinus mystax) with cardiomyopathy

Spontaneous intracardiac thrombosis is rarely reported in animals, particularly nonhuman primates. The finding of 2 cases of intracardiac thrombi in mustached tamarins (Saguinus mystax) that died as a consequence of congestive heart failure prompted us to do a retrospective study to determine the frequency of this condition. Clinical records, necropsy reports, and tissues from 60 mustached tamarins that died or were euthanized between 1996 and 2009 were reviewed. Of the 60 monkeys whose cases were reviewed, 10 (16.6%) had intracardiac thrombi, and 4 (6.6%) had dissecting aortic aneurysms. Of the 10 animals with intracardiac thrombosis, 3 had left ventricular involvement alone; 4 monkeys had thrombi only in the right ventricle, and the remaining 3 animals exhibited thrombi in both ventricles. Myocardial fibrosis and chronic renal disease were common findings in affected animals. The causes of the intracardiac thrombosis in the tamarins in the present study are not known, but the clinical signs and gross and microscopic lesions suggest that congestive heart failure secondary to cardiomyopathy is the primary contributor. In addition, the cause of the aortic dissecting aneurysms in the tamarins in this study is not known. Further studies are required to determine whether factors including aortic curvature, genetic background, or hypertension-alone or in combination-play a role. To our knowledge, the current retrospective study is the first report of intracardiac thrombosis and aortic aneurysms in mustached tamarins.     

The blood coagulation system as a molecular machine

The human blood coagulation system comprises a series of linked glycoproteins that upon activation induce the generation of downstream enzymes ultimately forming fibrin. This process is primarily important to arrest bleeding (hemostasis). Hemostasis is a typical example of a molecular machine, where the assembly of substrates, enzymes, protein cofactors and calcium ions on a phospholipid surface markedly accelerates the rate of coagulation. Excess, pathological, coagulation activity occurs in "thrombosis", the formation of an intravascular clot, which in the most dramatic form precipitates in the microvasculature as disseminated intravascular coagulation. Thrombosis occurs according to a biochemical machine model in the case of atherothrombosis on a ruptured atherosclerotic plaque, but may develop at a slower rate in venous thrombosis, illustrating that the coagulation machinery can act at different velocities. The separate coagulation enzymes are also important in other biological processes, including inflammation for which the rapid conversion of one coagulation factor by the other is not a prerequisite. The latter role of coagulation enzymes may be related to the old and probably maintained function of the coagulation machine in innate immunity.     

Clinicopathological features and the value of differential Cytokeratin 7 and 20 expression in resolving diagnostic dilemmas of ovarian involvement by colorectal adenocarcinoma and vice-versa

Introduction

Intravascular lesions of the hand comprise reactive and neoplastic entities. The clinical diagnosis of such lesions is often difficult, and usually requires pathologic examination. We present the largest series to date of intravascular lesions affecting the hand.

Methods

A retrospective review of intravascular (arterial and venous) lesions involving the hand was conducted. Data regarding clinicopathologic findings were analyzed.

Results

We identified 10 patients with intravascular lesions of their hands including thromboemboli (n = 3), reactive intravascular conditions such as papillary endothelial hyperplasia or Masson's tumor (n = 2) and fasciitis (n = 1), as well as vascular neoplasms including pyogenic granuloma (n = 2) and angioleiomyoma (n = 2).

Conclusion

Blood vessel injury and/or venous thrombosis may predispose to several intravascular lesions of the hand. Recognition of reactive entities from neoplastic conditions is important.     

Fibrinolytic activity in vascular grafts

We have demonstrated the absence of tissue fibrinolytic activator for 11 days following the transplantation of vascular grafts in dogs. The lack of a local tissue fibrinolytic activator (to dissolve intravascular thrombi) is suggested as an etiological factor in losses which occur during the first 11 days after digital replantation.     

Stabilizing Role of Platelet P2Y12 Receptors in Shear-Dependent Thrombus Formation on Ruptured Plaques

Background

In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y₁₂ receptors. However, such models do not predict involvement of P2Y₁₂ in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y₁₂ in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis.

Methodology

Plaques in the carotid arteries from Apoe ^−/− mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y₁₂ blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated.

Principal Findings

Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y₁₂ blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y₁₂ reduced the thrombi and increased embolization events. These P2Y₁₂ effects were present at shear rates >500 s⁻¹, and they persisted in the presence of coagulation. P2Y₁₂-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding.

Conclusions/Significance

Platelet P2Y₁₂ receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y₁₂ function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.     

Embolization itself stimulates thrombus propagation in pulmonary embolism

The role of active thrombosis in the pathophysiology of pulmonary embolism is unclear. We tested the hypothesis that venous thrombi significantly increase their thrombotic activity once they embolize into the high-flow circulation of the pulmonary arteries. Thrombotic activity was measured using an immunoassay that measures both fibrinopeptide B (FPB) as well as its most abundant metabolite des-arginine FPB. Thrombi were formed in the femoral veins of adult dogs. In one group, the thrombi were embolized without anticoagulation. In the second group, heparin (300 U/kg bolus, then 90 U x kg(-1) x h(-1) infusion) was administered before embolization to prevent subsequent thrombotic activity. Plasma FPB concentrations were significantly suppressed in the heparinized group relative to the nonheparinized group for 1 h postembolization (P = 0.038). We conclude that pulmonary embolization itself causes preexisting venous thrombi to greatly intensify their thrombotic activity and that embolization-associated thrombus propagation can be prevented by heparin.