Background: Hemodialysis (HD) grafts often fail due to stenosis at the venous anastomosis and thrombotic occlusion. Percutaneous management relies on thrombolysis with plasminogen activators, mechanical removal of thrombus and angioplasty of the stenotic lesion. Objectives: This report describes a phase I trial using Plasmin (Human) TAL 05-00018, a direct-acting fibrinolytic agent, to evaluate safety and, secondarily, to establish effective thrombolytic dosing. Patients/methods: Six cohorts of 5 patients with acute HD graft occlusion documented by angiography were treated with escalating dosages of plasmin (1, 2, 4, 8, 12, and 24 mg) infused over 30 minutes via criss-crossed pulse-spray catheters within the graft. The primary efficacy endpoint was >/=50% thrombolysis, as determined by comparison of pre- and 30-minute post-plasmin fistulograms. Results: Of 31 subjects who received study drug (safety population), 1 withdrew and 30 completed the trial (evaluable for efficacy). There was no significant change in plasma alpha-2 antiplasmin or fibrinogen concentration, major bleeding did not occur and there were no deaths. Serious adverse events in 4 patients were not related to study drug. There was a dose-response relationship for the primary efficacy endpoint, all 5 subjects receiving 24 mg achieving >75% lysis. Conclusions: This first phase I study of Plasmin (Human) TAL 05-00018, infused into thrombosed HD grafts, documents safety at dosages of 1 to 24 mg and an effective thrombolytic dosage of 24 mg. The results establish a foundation for further clinical study of catheter-based plasmin administration in thrombotic disorders.

P:URPOSE: To evaluate the safety and efficacy of a hydrodynamic thrombectomy system in a prospective, multicenter randomized comparison with pulse-spray thrombolysis in hemodialysis grafts. MATERIALS AND METHODS: Nine centers enrolled 120 adult patients with recently (</=14 days) thrombosed hemodialysis grafts. Graft venography was used to confirm occlusion in 62 patients randomly assigned to thrombectomy and 58 to thrombolysis. For thrombolysis, a mixture of 5,000 U of heparin and 250,000 U of urokinase was distributed throughout the thrombus, first to the venous then to the arterial graft end. For thrombectomy, the catheter was passed in the same sequence. Technical success was removal of 80% or more of thrombus. Clinical success was technical success plus the ability to dialyze. Also assessed were total procedure time, thrombus treatment time, procedure-related blood loss, other complications, and 30- and 90-day outcomes. RESULTS: Patient demographics were comparable. Technical success rates were 95%(59 of 62) for thrombectomy and 90%(52 of 58) for thrombolysis (P:=.31). Clinical success rates were 89%(55 of 62) and 81%(47 of 58), respectively (P:=.24). At 30 days, 69%(43 of 62) and 66%(38 of 58), respectively, could be dialyzed through the graft (P:=.70); at 90 days, the rates were 40%(25 of 62) and 41%(24 of 58), respectively (P:=.91). None of these differences or those for procedure-related blood loss and early and late complications were statistically significant. Thrombus treatment times of 16.8 minutes for thrombectomy and 23.4 minutes for thrombolysis were significantly different (P:<.01). CONCLUSION: The hydrodynamic thrombectomy system is at least as efficacious and safe as pulse-spray thrombolysis but shortens thrombus treatment time.

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Gd-DTPA was evaluated as a hepatic contrast agent for MR imaging. Twenty-six consecutive patients referred for suspected masses in the liver were studied at 1.5 T. Fourteen patients had hepatic metastases and one patient each had cholangiocarcinoma and multicentric hepatocellular carcinoma. Four patients had cavernous hemangiomas and the remainder had other benign lesions. Diagnoses were proved by biopsy, sonography, or radionuclide scintigraphy in 23 cases and by autopsy in one case. Precontrast scans were obtained by using standard pulse sequences. In addition, breath-hold scans were obtained before and after bolus administration of 0.1 mmol/kg Gd-DTPA by using a multislice T1-weighted gradient-echo pulse sequence with an ultrashort echo time. Mean lesion-liver signal difference/noise increased by 50%(p less than .01) in the immediate postcontrast phase. In two of 26 cases, multiple additional lesions as small as 3 mm were detected after contrast administration that were not seen before contrast administration. In no case was lesion-liver contrast worsened on scans obtained immediately after administration of contrast material. However, on delayed scans, detection of lesions worsened in some cases because of equilibration of contrast material between liver and lesion. These initial clinical results suggest that enhancement with Gd-DTPA is a practical method for improving lesion-liver contrast and has the potential to improve the accuracy of MR imaging in the liver. However, optimized fast imaging techniques are required for best results.

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions"(ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1,-4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1,-4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.

BACKGROUND: Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. METHODS: Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. RESULTS: A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. CONCLUSIONS: Systemic coagulation disturbances are associated with delayed xenograft rejection.

BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.

Directional atherectomy alone or with supplemental percutaneous transluminal angioplasty was used to treat peripheral vascular lesions in 77 patients (85 procedures). Lesions involved 17 iliac arteries, 45 infrainguinal arteries, and 23 laser extremity vein bypass grafts. Technical success, defined as reduction of stenosis diameter to 30% or less of the normal vessel diameter, was achieved in 78 of 85 (92%) cases. The complication rate was 21%(18 of 85 procedures). Most complications were minor and were related to puncture sites. Patients underwent noninvasive follow-up studies, including measurement of ankle-brachial index and segmental pressures, plethysmography, and clinical examination. The mean follow-up period was 13.5 months. The probability of 1-, 2-, and 3-year patency for lesions treated with atherectomy alone was 92%, 84%, and 84%, respectively. Kaplan-Meier survival analysis revealed no difference in 2- to 3-year patency rate on the basis of lesion location or presence of calcification, eccentricity, or ulceration. Diabetic patients, however, had a higher restenosis rate than did patients who were not diabetic (P less than .03).

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.