Ascites-induced LeVeen shunt coagulopathy.

Ten of 11 patients undergoing peritoneovenous (LeVeen) shunt placement for intractable ascites had disseminated intravascular coagulation (DIC) following the shunt procedure. Intraoperative ascitic fluid specimens revealed fibrin split products (FSP) in high titer (1:100-1:1600) in all patients. Endotoxin was found in 6 of 11 ascitic fluid samples but in no plasma samples. Activated clotting factors, clot inhibitors, excess protein, and fibrinolytic activity were not found in ascitic fluid. Clotting factor levels were much lower than in plasma. Bleeding occurred after operation in two patients; this appeared to be related to the severity of liver dysfunction as demonstrated by elevations of bilirubin, serum glutamic oxalocetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and preoperative DIC. It is concluded that the LeVeen shunt coagulopathy is DIC, and may be related to exposure of the systemic circulation to FSP-rich ascitic fluid that may activate the coagulation mechanism. Bleeding complications do not appear to be related to the severity of the post shunt coagulopathy, but rather to the severity of liver dysfunction and presence of preoperative DIC (probably caused by the liver disease).     

Compound 48/80 suppresses monocytic tissue factor-initiated extrinsic blood coagulation induced by bacterial endotoxin

BACKGROUND: Hypercoagulability is one of the commonly exhibited endotoxemia septic symptoms; it could contribute to the manifestation of disseminated intravascular coagulation presenting threats to cardiovascular functions. The underlying mechanism, however, remains largely complex and unknown. OBJECTIVES: We herein determine whether bacterial endotoxin (LPS) upregulates the activities of clotting factors in plasma, contributing to extrinsic hypercoagulation. Compound 48/80 (48/80) is also tested for its ability to suppress hypercoagulation. METHODS: In an in vitro infection model, we exposed whole blood to LPS (Escherichia coli 0111:B04; 100 ng/ml) for 2 h. Thrombin time (TT), prothrombin time (PT), and the activities of clotting factors ( FVII, FIX, FX ) in plasma contributing to the extrinsic coagulation were determined. Peripheral blood monocytes were isolated from Histoplaque 1077 gradient centrifugation, and the procoagulant activity was determined by a single-stage clotting assay on a Fibrometer. RESULTS: LPS drastically activated monocytic procoagulant activity which was defined as tissue factor (TF) activity, whereas LPS had no effect on TT, PT, and the activities of clotting factors in plasma. 48/80 not only instantaneously offset LPS-induced monocytic TF activation, but also significantly inhibited PT including the activities of clotting factors (FVII, FIX, and FX) in plasma, whereas TT remained unaffected. CONCLUSIONS: Monocytic TF activation was solely responsible for the extrinsic hypercoagulation in response to LPS. 48/80 effectively suppressed LPS-induced monocyticTF-initiated extrinsic coagulation at multiple sites, possibly presenting a new therapy for an instantaneous relief of hypercoagulation under septic conditions.     

In vitro effects of detergent sclerosants on coagulation, platelets and microparticles.

OBJECTIVES: To investigate the in vitro effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on clotting tests, clotting factors, platelets and microparticles. MATERIALS AND METHODS: Platelet rich (PRP) and platelet poor (PPP) plasmas were incubated with varying concentrations of STS and POL. Clotting tests, platelet/plasma turbidity, and microparticle studies were performed. Specimens were mixed with individual factor deficient plasmas and clotting factor activities were studied. RESULTS: STS at high concentrations (>0.3%) destroyed platelets, microparticles and the clotting factors V, VII and X. It prolonged all clotting tests including prothrombin time (PT), activated partial thromboplastin time (APTT), non-activated partial thromboplastin time (NAPTT), thrombin time (TT), factor Xa clotting time (XACT) and surface activated clotting time (SACT). Higher concentrations of POL were required to achieve some anticoagulant activity. Low sclerosant concentrations shortened XACT and SACT, and induced release of procoagulant platelet derived microparticles. Increased exposure time resulted in increased procoagulant activity. STS at concentrations higher than 0.5% precipitated a complex containing apolipoprotein b and fibrinogen. CONCLUSIONS: Detergent sclerosants affect the clotting mechanism by interfering with clotting factor activities, procoagulant phospholipids and platelet derived microparticles. STS has more anticoagulant activity than POL in high concentrations. Low concentration sclerosants demonstrate procoagulant activity.     

Procoagulant activity of ascitic fluid in hepatic cirrhosis: in vivo and in vitro.

Three cases of disseminated intravascular coagulation following infusion of ascitic fluid from the peritoneal cavity to the vascular system are reported in patients with hepatic cirrhosis. One of these was treated with intermittent drainage and infusion, and in the other two a LeVeen shunt was used. Studies on the ascitic fluid indicate the presence of a procoagulant material which would appear to be an activator of factor X. Preactivated factor X (Xa) from the fluid and/or a tissue factor activator of factor VII are additional possibilities.     

Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation

Miwa Y, Yamamoto K, Onishi A, Iwamoto M, Yazaki S, Haneda M, Iwasaki K, Liu D, Ogawa H, Nagasaka T, Uchida K, Nakao A, Kadomatsu K, Kobayashi T. Potential value of human thrombomodulin and DAF expression for coagulation control in pig‐to‐human xenotransplantation. Xenotransplantation 2010; 17: 26–37. © 2010 John Wiley & Sons A/S. Abstract: Background: Problems of coagulation disorder remain to be resolved in pig‐to‐primate xenotransplantation. Molecular incompatibilities in the coagulation systems between pigs and humans, such as the thrombomodulin (TM)‐protein C system or direct prothrombinase activity, have been suggested as possible causes. Coagulation and complement activation are closely related to each other. The purpose of this study was to elucidate the protective effects on the coagulation system of the expression of human TM and decay accelerating factor (hDAF) (for inhibition of complement activation) in pig endothelial cells. Methods: Human aortic endothelial cells (HAEC), porcine aortic endothelial cells (PAEC), hDAF‐expressing PAEC (hDAF‐PAEC), hDAF/Endo‐β‐galactosidase C‐expressing PAEC (hDAF/EndoGalC‐PAEC), hTM‐expressing PAEC (hTM‐PAEC), hDAF/hTM expressing‐PAEC (hDAF/hTM‐PAEC), and hDAF/EndoGalC/hTM‐expressing PAEC (hDAF/EndoGalC/hTM‐PAEC) were used in this study. Coagulation activity was examined by clotting, activated protein C (APC), and thrombin generation assay. Results: A large difference was observed in clotting time of human plasma when exposed to PAEC (170 s) and HAEC (1020 s). hTM expression on PAEC was proven to produce a comparable level of APC to that produced by HAEC, which prolonged the clotting time, though not to the level of HAEC. Pretreatment with human sera considerably shortened the clotting time in PAEC (80 s). hDAF‐PAEC significantly inhibited such a shortening of clotting time by reductions in tissue factor expression and thrombin generation. Thrombin generation through direct prothrombinase activity, which was detected only in PAEC, could be suppressed by hTM expression. Suppression of antibody binding and complement activation improved clotting time not in PAEC, but in PAEC expressing hTM. Conclusions: In addition to effective suppression of antibody‐induced complement activation, hTM expression in PAEC may be essential for regulating procoagulant activity in xenotransplantation.     

Coagulopathy post peritoneovenous shunt.

In 1942, 53% of medically treated patients with cirrhosis were dead 6 months after the onset of ascites. Only 30% survived 1 year. This dismal outlook has improved only slightly with advances in medicine. Yet, some internists reject the peritoneovenous shunt (PVS) for this fatal condition even if they are aware that a diminished blood volume causes the abnormal sodium retention responsible for ascites. Their objections are based on life-threatening complications of PVS, especially post shunt coagulopathy (PSC). Blood shed into the peritoneal cavity becomes incoagulable. Such blood is immediately coagulated by a protocoagulant (soluble collagen) and concurrently lysed by tissue plasminogen activator (TPA) secreted by the peritoneal serosa. Wide zones of lysis surround peritoneal tissue placed on fibrin plates. Large volumes of ascitic fluid infused into circulating blood simulates the fate of blood shed into the peritoneal cavity with lysis playing the major role. Addition of ascitic fluid to normal platelet-rich plasma in vitro initiates clot lysis on thromboelastogram (TEG). Epsilon-aminocaproic acid (EACA) counteracts this lysis. EACA and clotting factors normalize the TEG and arrest PSC. Disposal of ascitic fluid at surgery prevents or ameliorates PSC. Mild PSC was encountered only twice in 150+ consecutive patients (1.3%) with only one case being clinically significant (0.6%). Severe PSC occurred seven times in 98 early shunt patients whose ascitic fluid was not discarded. Severe PSC requires shunt interruption and control of bleeding with clotting factors and EACA. Peritoneal lavage with saline prevents the recurrence of PSC on reopening the shunt. In four patients, EACA and clotting factors were adequate to arrest coagulopathy. Three earlier patients died of PSC before its cause and treatment were understood. Proper management eliminates this life-threatening complication, and PSC cannot be considered a deterrent to PVS. Disseminated intravascular coagulopathy (DIC) is produced in experimental animals only by the injection of thrombin or thromboplastin. PSC is a distinct entity differing from DIC; EACA and not heparin is the antidote for PSC.     

Malignant ascites. Clinical and experimental observations.

Malignant ascites formation is a grave prognostic sign, but palliative efforts seem justified in some patients. Lack of knowledge concerning the natural history of this process hinders the choice of therapeutic options. Over 5 years, 107 patients with untreated malignant ascites were reviewed to define their survival. Pancreas (20), ovary (18), and colon (18) were the most frequent tumors, with 52% of patients presenting with ascites at the time of the initial cancer diagnosis. Cytology evaluation of the ascitic fluid was positive for tumor cells in 57% of cases and a high protein content was noted in 65%. Mean survival of the entire series was only 20 weeks from the time of diagnosis of ascites, with tumors of ovarian and lymphatic origin having better mean survivals of 32 and 58 weeks, respectively. Patients with high ascitic protein levels fared better than those with low levels. In an effort to explain this correlation of elevated protein levels and a favorable survival rate, a hypothesis was proposed that certain tumors secrete a factor, which alters vascular permeability and causes fluid accumulation in the absence of lymphatic obstruction. In an experimental rat model of malignant ascites, the intraperitoneal infusion of cell-free malignant ascitic fluid caused an increase in edema formation and a significant increase in capillary permeability to protein in the omentum. This demonstrated change in the leak of protein explains the formation of ascites by some tumors in the absence of tumor obstruction of the draining lymphatics of the peritoneal cavity and suggests another important mechanism in the genesis of malignant ascites.     

Microparticles during sepsis and trauma. A link between inflammation and thrombotic processes

Sepsis and trauma lead to a sustained activation of monocytes and endothelium. In the vascular compartment, stimulated cells release microparticles. Circulating MP provide an additional procoagulant phospholipid surface enabling the assembly of the clotting enzymes complexes and thrombin generation. Their procoagulant properties rely on the exposition of phosphatidylserine, made accessible after cell stimulation and on the possible presence of tissue factor, the main cellular initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor activity. At sites of endothelium injury, enhanced release or recruitment of procoagulant MP through P-selectin-PSGL-1 pathway could concentrate TF activity above a threshold allowing blood coagulation to be triggered. Converging evidences from experimental or clinical data highlight a role for MP harboring tissue factor in the initiation of disseminated intravascular coagulopathy. In these settings, the pharmacological modulation of MP levels or biological functions through activated protein C or factor VIIa allows challenging issues.     

Increased human tumor necrosis factor-α levels induce procoagulant change in porcine endothelial cells in vitro

Lee KG, Lee H, Ha JM, Lee YK, Kang HJ, Park C‐G, Kim SJ. Increased human tumor necrosis factor‐α levels induce procoagulant change in porcine endothelial cells in vitro. Xenotransplantation 2012; 19: 186–195. © 2012 John Wiley & Sons A/S. Abstract: Background: Intravascular thrombosis and systemic coagulation abnormalities are major hurdles to successful xenotransplantation and are signs of acute humoral rejection. Increased expression of tissue factor (TF) is associated with the development of microvascular thrombosis in xenografts. To develop an effective strategy to prevent accelerated coagulation in xenografts, we investigated the mechanism by which porcine endothelial cells (PECs) become procoagulant after contact with human blood. Methods: The changes in TF mRNA levels and activity in PECs after incubation with 20% human serum or human bioactive molecules, including C5a, tumor necrosis factor‐α (TNFα) and interleukin (IL)‐1α, were evaluated using real‐time PCR and the factor Xa chromogenic assay, respectively. The procoagulant changes in PECs by these agonists were evaluated by measuring the coagulation time of human citrated plasma suspended with PECs pretreated with each agonist. TF expression and coagulation times were also assessed in PECs transfected with short interfering RNA (siRNA) designed to knock down porcine TF. We also examined the production of proinflammatory cytokines in human whole‐blood or plasma after contact with PECs, which were screened using the cytometric bead array system. TNFα levels were measured using ELISA in whole‐blood after contact with PECs, with or without the addition of xenoreactive antibodies or C1 esterase inhibitor. Results: Porcine TF mRNA and activity in PECs were up‐regulated in response to human TNFα and IL‐1α but were not affected by C5a or 20% human serum. Up‐regulation of TF expression by human TNFα or IL‐1α shortened PEC‐induced coagulation time, while siRNA‐mediated knockdown of TF expression prolonged coagulation time. The incubation of PECs with human whole‐blood led to a significant increase in human TNFα levels in the blood, which was promoted by the addition of xenoreactive antibodies and prevented by C1 esterase inhibitor. Conclusions: Human TNFα level increases in human blood after contact with PECs, which is attributed to xenoreactive antibody binding and subsequent complement activation. Human TNFα induces procoagulant changes in PECs with increased TF expression. This study suggests that human TNFα may be one of the mediators linking complement activation with procoagulant changes in the xenoendothelium.     

Fresh plasma and concentrates of clotting factors for therapy of perioperative coagulopathy: what is known?

Acquired, perioperative coagulopathy often develops due to acute bleeding. In the case of primarily healthy patients with normal bone marrow and liver functions, a lack of coagulation factors initiates coagulopathy before secondary thrombopenia arises. Replacement of coagulation factors can be performed by infusion of fresh plasma (single donor or pooled plasma) or concentrates of clotting factors. Fresh plasma as well as concentrates of clotting factors available in German-speaking countries are of high quality and fulfil all safety standards. Undesirable side-effects due to transmission of infections and immunological reactions are--in all probability--more uncommon for virus-inactivated plasma and clotting factors than for single donor plasma. In contrast, thromboembolic complications are unlikely when using fresh frozen plasma, because it contains a balanced ratio of pro-coagulatory and anti-coagulatory factors. For virus-inactivated pooled plasma and concentrates of clotting factors, sporadic reports of thromboembolic events have been published. Concentrates of clotting factors can be stored easily and are rapidly prepared for use. In contrast, fresh frozen plasma has to be thawed before application leading to a significant delay in the schedule. During activated hemostasis, the half-life of clotting factors is significantly reduced in comparison to a stable physiological situation. In the case of perioperative coagulopathy higher dosages of fresh plasma and clotting factors than those recommended in published guidelines are often necessary for successful treatment. When using fresh plasma for coagulation therapy the resulting volume load must be considered. In conclusion, a modern concept of perioperative coagulation management should include fresh plasma as well as concentrates of clotting factors. The anesthetist should be familiar with the available components and be able to consider and adapt them to the individual situation.     

Frischplasma und Faktorenkonzentrate zur Therapie der perioperativen Koagulopathie

Acquired, perioperative coagulopathy often develops due to acute bleeding. In the case of primarily healthy patients with normal bone marrow and liver functions, a lack of coagulation factors initiates coagulopathy before secondary thrombopenia arises. Replacement of coagulation factors can be performed by infusion of fresh plasma (single donor or pooled plasma) or concentrates of clotting factors. Fresh plasma as well as concentrates of clotting factors available in German-speaking countries are of high quality and fulfil all safety standards. Undesirable side-effects due to transmission of infections and immunological reactions are – in all probability – more uncommon for virus-inactivated plasma and clotting factors than for single donor plasma. In contrast, thromboembolic complications are unlikely when using fresh frozen plasma, because it contains a balanced ratio of pro-coagulatory and anti-coagulatory factors. For virus-inactivated pooled plasma and concentrates of clotting factors, sporadic reports of thromboembolic events have been published. Concentrates of clotting factors can be stored easily and are rapidly prepared for use. In contrast, fresh frozen plasma has to be thawed before application leading to a significant delay in the schedule. During activated hemostasis, the half-life of clotting factors is significantly reduced in comparison to a stable physiological situation. In the case of perioperative coagulopathy higher dosages of fresh plasma and clotting factors than those recommended in published guidelines are often necessary for successful treatment. When using fresh plasma for coagulation therapy the resulting volume load must be considered. In conclusion, a modern concept of perioperative coagulation management should include fresh plasma as well as concentrates of clotting factors. The anesthetist should be familiar with the available components and be able to consider and adapt them to the individual situation.     

Infant cardiopulmonary bypass: a procoagulant state.

BACKGROUND: Procoagulant activity after cardiopulmonary bypass (CPB) in infants may predispose to thrombotic and bleeding complications. The induction of tissue factor and prothrombinase activity on endothelial cell membranes is a primary step in the activation of the extrinsic clotting cascade. The purpose of this study is to characterize the fibrinolytic and endothelial procoagulant state in infants undergoing congenital cardiac repairs with and without CPB. METHODS: Fourteen infants (aged 1 to 12 weeks) underwent repair of congenital cardiac defects. Two patients had closed procedures (controls) and 12 had open cardiac procedures. Serum samples were taken before and after CPB, 1, 4, and 24 hours after CPB. Tissue plasminogen activator, plasminogen activator inhibitor-1, interleukin-1beta, interleukin-6, plasma tissue factor, and factor V levels were measured. Human umbilical vein endothelial cell cultures were incubated with serum taken from the above time points and assayed for induction of tissue factor and prothrombinase activity. RESULTS: Control patients had no change from preoperative values in any of the parameters examined. In experimental patients, tissue plasminogen activator levels peaked at 1 hour after CPB and then decreased to normal by 24 hours. Plasminogen activator inhibitor-1 levels peaked at 4 hours after CPB and returned to baseline by 24 hours. The plasma of all patients had no intrinsic tissue factor activity. Induction of tissue factor activity on umbilical vein endothelial cells peaked immediately and again at 24 hours, whereas prothrombinase activity peaked early and stayed elevated. Serum factor V levels were significantly reduced after CPB, but returned to near baseline levels by 24 hours. CONCLUSIONS: Cardiopulmonary bypass is associated with derangement of the coagulation and fibrinolytic systems in infants. The serum of these patients promotes the induction of endothelial procoagulant activity, suggesting that there may be a hypercoagulable state in the postbypass period.     

Fresh frozen plasma supplement to massive red blood cell transfusion.

The efficacy of supplemental fresh frozen plasma (FFP) therapy after massive packed red cell (PRBC) replacement for hemorrhagic shock was studied in 22 conditioned dogs. Ten dogs were randomized to received FFP, balanced electrolyte solution (BES), and PRBC, while 12 dogs received BES and PRBC. Coagulation factor activity for Factors I, II, V, VII and VIII, as well as antithrombin III (AT III), prothrombin time, partial thromboplastin time, and thrombin time, were measured at preshock, postshock, postresuscitation, and postshock day two. All coagulation factor activities fell with shock and decreased further with resuscitation in both groups. Factor II (a procoagulant) and AT III (an anticoagulant) fell significantly less after resuscitation in the plasma dogs; otherwise, no postresuscitation differences were seen. The changes in factor activity from postresuscitation until day two reflected factor half life and molecular weight, independent of FFP therapy. These data show that prophylactic FFP therapy does not efficiently restore coagulation activity. Consequently, routine FFP therapy for its procoagulant effects after hemorrhagic shock should be abandoned pending controlled studies in man.     

Haemostaseological aspects of perioperative blood management

Recent studies in humans have shown that tissue factor on the surface of endothelial cells, monocytes, or subendothelial structures sparks plasmatic coagulation. In vivo, there is no functional separation of an "endogenous" and "exogenous" pathway of the coagulation cascade. However, global laboratory tests run along such pathways due to preincubation with specific activators and, hence, allow localization of inherited coagulation defects. Coagulation inhibitors such as antithrombin or activated protein C are accelerated in their activity by cell surface glycoproteins and almost completely inactivate procoagulant activity in the microcirculation. Antithrombin binds to endothelial glycosaminoglycans and then significantly increases anticoagulant activity. Protein C is activated by the thrombin-thrombomodulin-complex and inactivates factors V a and VIII a, respectively. Additionally, activated protein C has a profibrinolytic effect. Both systems physiologically counteract the procoagulant transformation of endothelial and monocyte cell surfaces which occurs in critically ill patients due to induction of tissue factor, suppression of thrombomodulin, and removal of glycosaminoglycans from the cell surface. The distinction of static and dynamic coagulation disorders is useful since static disorders seldom require therapeutic interventions although global laboratory tests may continuously deteriorate. Dynamical disorders are symptoms of an underlying disease, and consumption coagulopathy with disseminated fibrin deposition and oozing occurs when coagulation turnover cannot be stopped. Antithrombin substitution is a well documented therapeutic option along with fresh frozen plasma and erythrocyte concentrate transfusion for blood substitution. Recent case reports in patients with irreversible bleeding complications favour the application of a recombinant factor VII concentrate. A rising perspective to decrease the use of heterologous blood and blood products may be intraoperative plasma retransfusion. The quality of such plasma undergoing consecutive filtration steps has to be clinically studied. The application of a synthetic platelet substitute, the "plateletsome", containing platelet glycoproteins led to significantly improved haemostasis without generating systemic procoagulant activity. In a far future, procoagulant cell surface transformation may be influenced by topic application of inhaled thrombomodulin loaded liposomes or by sense or antisense oligonucleotides inducing thrombomodulin expression or suppressing tissue factor expression, respectively.     

Determination of hypercoagulable state in acute bronchospasm.

The issue of hypercoagulability in acute asthmatic attacks is controversial. This may be due to lack of an appropriate test to monitor overall coagulation. Current hematologic tests do not account for the cellular fraction of blood that has procoagulant activity. Our study uses a clotting assay called the modified recalcification time test that is performed with whole blood to ensure the contribution of all chemical and cellular mediators in the coagulation process, particularly tissue factor. Venous blood samples were obtained from 12 adult patients with acute exacerbation of asthma or chronic obstructive pulmonary disease and compared with samples from 12 age-matched healthy control subjects. By use of the modified recalcification time, the presence of a relative hypercoagulable state was demonstrated in patients with acute bronchospasm. Furthermore, there is an identifiable difference in modified recalcification time value between the patients with acute attacks who required hospital admission versus those discharged from the emergency department.     

Usefulness of a Nonmachinery Based System for the Reinfusion of Cell-Free and Concentrated Autogenous Ascitic Fluid

Abstract: A nonmachinery based system using gravity dependent flow for the treatment and reinfusion of ascitic fluid was developed, and its usefulness was assessed. In a preliminary study using bovine plasma, samples with protein concentrations below 5.0 g/dl were found to be treatable with this system. Bovine plasma containing blood, prepared to 0.5% hematocrit and with a protein concentration of 3.0 g/dl, was also treatable. We conducted a clinical study of 1,799 treatment sessions (1,495 using a machinery based system and 304 using a nonmachinery based system) of 343 patients with ascites refractory to various treatments. The recovery ratio of protein from the original ascitic fluid was 96% using the nonmachinery based system and 77% with the machinery based system (p < 0.01). Of 253 continuous reinfusions of ascitic fluid using the nonmachinery based system, the original ascitic fluid at protein concentrations below 2.5 g/dl was treatable. Original ascitic fluid below a hematocrit of 0.7% (protein concentration, 1.4 g/dl) was also treatable. This new procedure was simple and time and labor saving; the high recovery ratio of protein also demonstrated the usefulness of the new system.     

Clue to diagnosis of neonatal urinary ascites. Relative radioluency of liver shadow

About 25 per cent of neonatal ascites is caused by urinary tract disease. Opacification of the ascitic fluid by leakage of contrast material during cystography or intravenous urography may lead to striking relative radioluency of the liver. This phenomenon may be an important clue to the differential diagnosis of the etiology of neonatal ascites.     

Procoagulant activity in normal human urine associated with subcellular particles

Procoagulant activity (PCA) in normal human urine was found to be sedimented by centrifugation at X 100,000g. Therefore, studies were done to identify the structures associated with the procoagulant activity. Transmission electron microscopy of the X 100,000g pellet revealed numerous membrane-bound vesicles as well as fibrous material. Filtration of normal urine through a 0.2-micron filter removed more than 90% of the procoagulant activity. Scanning electron microscopy of the filter surface revealed 0.1 to 1.1 micron particles and fibrous material. By centrifugation at pH 3 and 5 the fibrous material and particles were separated. The procoagulant activity remained with the particles in each case. The fibrous material was shown to be Tamm-Horsfall protein by SDS-PAGE and Western blotting using anti-Tamm-Horsfall protein serum. Purified Tamm-Horsfall protein itself was not procoagulant. Therefore, PCA in normal human urine is associated with particles 0.1 to 1.1 micron in diameter which appear to be lipid membranes in various arrangements.