Direct microscopic monitoring of initial and dynamic clot lysis using plasmin or rt-PA in an in vitro flow system.

INTRODUCTION: Plasmin is a direct-acting thrombolytic agent with a favorable safety profile upon intra-arterial delivery in pre-clinical and phase I studies. However, the thrombolytic efficacy of plasmin, relative to that of rt-PA, remains to be established. We have compared the dynamics of clot lysis with plasmin or rt-PA in an in vitro perfusion system, in which thrombolytic agent is administered locally, allowed to induce lysis for short intervals, then washed with plasma in a re-circulation circuit. MATERIALS AND METHODS: Whole blood human clots were prepared in observation chambers, exposed to plasmin or rt-PA at equimolar concentrations (1.2/1.0, 1.8/1.5 and 2.4/2.0 mg/ml) for measured intervals of time, followed by perfusion with human plasma. Clot size was monitored by digital analysis of sequential photographs obtained through an optical microscope. RESULTS: Plasma perfusion after incubation with thrombolytic agent rapidly removed superficial clot fragments. This initial decrease in clot size was greater with plasmin than with rt-PA when tested at the highest concentrations of agent (0.63 ± 0.11 vs. 0.30 ± 0.11, p=0.001 for clots with non-cross-linked fibrin and 0.53 ± 0.15 vs. 0.14 ± 0.15, p=0.02, for clots with cross-linked-fibrin). Subsequent clot lysis during plasma flow was greater after prior incubation with rt-PA. Longer incubation times of plasmin resulted in larger portions of the clot being washed free. Repeated plasmin incubations and plasma perfusions of a clot successfully induced stepwise reductions in clot size. CONCLUSIONS: Initial clot lysis is greater with direct exposure using plasmin than rt-PA. During washout and circulation with plasma, rt-PA induced continued clot lysis, while plasmin lysis was curtailed, presumably because of plasmin inhibition.

Comparison of local thrombolytic efficacy of plasmin and rt-PA in an in-vitro flow system; a pilot study.

Plasmin, a directly acting thrombolytic agent, demonstrated a very favorable safety profile upon intra-arterial delivery to the clot site; however, its thrombolytic efficacy remains to be further assessed. In this study, differences in thrombolysis between clots exposed to equimolar concentrations of plasmin and recombinant tissue-type plasminogen activator (rt-PA) after partial vessel recanalization were tested in a model system. Model blood clots were prepared in glass chambers enabling direct observation by dynamic optical microscopy. The incubation of clots with plasmin (2.4 mg/ml) or rt-PA (2.63 mg/ml), allowing for the initial biochemical clot degradation, was followed by 'flushing' the clots with tangentially directed plasma flow devoid of a thrombolytic agent, mimicking blood flow after partial vessel recanalization. The acquired images were analyzed for nondissolved blood clot area as a function of time. With both thrombolytic agents, the relative clot area decreased rapidly in the first 30 s after initiation of perfusion due to 'flushing' the degraded clot fragments (after plasmin by 0.26 ± 0.22 and after rt-PA by 0.34 ± 0.21, P = 0.60). In the following minutes, the clot size showed a linear time dependence: after incubation with plasmin the clot size did not change substantially any more, whereas after incubation with rt-PA the clot size continually decreased. The slopes of the regression lines differed significantly (r(pl) = -8.9 10 vs. r(rtPA) = -44.1 10/min, P < 0.01). In conclusion, the thrombolytic action of plasmin was terminated rapidly by contact with flowing blood plasma, whereas the thrombolytic action of rt-PA was prolonged.

Interpretation of endpoints in a network meta-analysis of new oral anticoagulants following total hip or total knee replacement surgery.

New oral anticoagulant (NOAC) regimens [dabigatran 150 mg (D150) and 220 mg (D220), rivaroxaban 10 mg (R20), and apixaban 2.5 mg bid (A5)] were effective and safe compared to enoxaparin for the prevention of venous thromboembolism (VTE) following elective total knee (TKR) or hip replacement (THR) surgery. First a cluster analysis was used to identify homogeneous studies for the trial programs of each NOAC. Second, only studies reporting VTE and VTE-related death, major bleeding, and mortality were included. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each NOAC regimen versus the comparator. Third, these data were used for the indirect comparison between NOACs. Cluster analysis identified duration of treatment (10 ± 5 and 34 ± 5 days) as the only homogeneous parameter across all NOAC programs (p>0.05) except for A5 and VTE over 10 ± 5 days (analysis not performed). The results of the calculated OR and 95% CI of the four NOAC regimens over 10 ± 5 and 34 ± 5 days showed inferiority of D150 and D220 compared to R10 for VTE (p<0.01, p<0.001). Comparisons of major bleeding and mortality were not different for all indirect comparisons. Despite the lack of standard definitions for VTE and bleeding outcomes, cluster analysis seems to be an appropriate tool to identify homogeneity across trial programs and to perform an indirect comparison for NOACs for prevention of VTE following TKR and THR surgery.

Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke.

BACKGROUND: Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat. METHODS: Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm. RESULTS: The main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2-3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats. CONCLUSIONS: The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.

Interference of thrombin in immunological assays for hirudin specific antibodies.

Recombinant hirudins (desirudin, lepirudin) are direct thrombin inhibitors administered as anticoagulants for heparin-induced thrombocytopenia (HIT) and venous thromboembolism (VTE) prophylaxis. Although these small polypeptides are widely used, concern exists over reports of antigenicity. In the largest study of r-hirudin immunogenicity to-date, we evaluated the prevalence, quantity and specificity of IgG immune responses to desirudin (15 mg SC q12h for as long as clinically required) in 245 surgical and medically-ill subjects enrolled in DESIRABLE, a multicenter, open-label, clinical trial of hospitalized patients requiring VTE prophylaxis. Sera obtained before and 30 days after desirudin administration were analyzed for IgG anti-desirudin by immunoenzymetric assay using immobilized desirudin to bind desirudin-reactive antibody and peroxidase conjugated monoclonal-anti-human IgG Fc to detect bound IgG antibody. Of 245 study subjects, 19 (7.7%) were antibody "responders" (>2-fold increase in IgG antibody levels with >50% inhibition by desirudin 30 days post-treatment). There were no differences between responders and non-responders in incidence of clinical outcomes or bleeding-related adverse events. Forty-six patients had detectable desirudin-reactive IgG antibody prior to treatment, with no significant increase in antibody levels after exposure and no increase in clinical events. The origin of pre-existing hirudin-reactive IgG antibody requires further investigation involving suspected anti-thrombin-thrombin interactions. These results indicate a low potential for immunogenicity, with <8% of patients developing IgG antibodies after desirudin administration for VTE prophylaxis. In contrast to reports on lepirudin, production of anti-hirudin antibodies to desirudin has no apparent effect on clinical events.

Glomerular hemophagocytic macrophages in a patient with proteinuria and clinical and laboratory features of hemophagocytic lymphohistiocytosis (HLH).

Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous disorder characterized by excessive activation and proliferation of nonmalignant histiocytes, which are commonly found in bone marrow, lymph nodes, spleen and liver in affected patients. Here, we report the presence of glomerular macrophages, including one showing erythrophagocytosis, on renal biopsy in a 25-year-old patient with clinical presentation and laboratory changes consistent with HLH. The clinical course was marked by persistent fever for 2 months, pleural and pericardial effusion, splenomegaly, lymphadenopathy, pancytopenia, cardiac arrhythmias, multiple organ dysfunction, and proteinuria, with demise after a 2-month hospitalization. Positive assay for Epstein-Barr virus (EBV), marked hyperferritinemia, hypofibrinogenemia, hypertriglyceridemia, elevated anti-nuclear antibody, proteinuria, and decreased circulating NK cells by flow cytometry were compatible with the diagnosis of HLH. We suggest that the glomerular hemophagocytic macrophages, which have not heretofore been described in the kidney of a patient with HLH, may have contributed to renal dysfunction manifest as proteinuria.

Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model.

BACKGROUND: Intra-arterial (IA) administration of rt-PA for ischemic stroke has the potential for greater thrombolytic efficacy, especially for a large thrombus in the M1 or M2 segment of the middle cerebral artery (MCA). Intracranial hemorrhage (ICH) is a concern with IA or intravenous (IV) administration especially as the therapeutic window is extended. However, because IA administration delivers a higher local concentration of agent, the incidence and severity of ICH may be greater than with similar doses IV. We investigated the safety of rt-PA administration by IA compared to IV infusion following 6 hours of MCA occlusion (MCAo) with reflow in the spontaneously hypertensive rat (SHR). METHODS: Male SHRs were subjected to 6 hours MCAo with 18 hours reflow using a snare ligature model. They were treated with IA saline, IA rt-PA (1, 5, 10, 30 mg/kg), or IV rt-PA (10 and 30 mg/kg) by a 10 to 60 minute infusion beginning approximately 1 minute before reflow. The rats were recovered for 24 hours after MCAo onset at which time Bleeding Score, infarct volume, and Modified Bederson Score were measured. RESULTS: Greater hemorrhagic transformation occurred with 10 and 30 mg/kg rt-PA administered IA than IV. The IV 10 mg/kg rt-PA dosage induced significantly less bleeding than did the 1 or 5 mg/kg IA groups. No significant increase in infarct volume was observed after IA or IV treatment. Rats treated with 30 mg/kg rt-PA by either the IA or IV route had greater neurological dysfunction compared to all other groups. CONCLUSIONS: Administration of rt-PA by the IA route following 6 hours of MCAo results in greater ICH and worse functional recovery than comparable dosages IV. Significantly greater bleeding was observed when the IA dose was a tenth of the IV dose. The increased bleeding did not translate in larger infarct volumes.

Ancrod and fibrin formation: perspectives on mechanisms of action.

BACKGROUND AND PURPOSE: Ancrod, derived from Malayan pit viper venom, has been tested as ischemic stroke treatment in clinical trials with inconsistent results. We studied the actions of ancrod on fibrinolysis pathways in patient plasma samples and endothelial cell culture systems. METHODS: We analyzed fibrinogen levels during the first 6 hours of ancrod infusion in patients entered in the Stroke Treatment with Ancrod Trial. For the in vitro study, human brain microvascular endothelial cells incubated with plasminogen or with human brain microvascular endothelial cell-conditioned medium were co-incubated with ancrod and fibrinogen under normal or oxygen-glucose deprivation conditions over 6 hours. RESULTS: Fibrinogen levels decreased both in vivo and in vitro. Ancrod generated fibrinopeptide A, caused visible clot formation, and reduced levels of tissue-type plasminogen activator antigen in the human brain microvascular endothelial cell system and in a cell-free system with conditioned media. CONCLUSIONS: The in vitro results indicate that ancrod causes local fibrin formation and secondary depletion of tissue-type plasminogen activator by binding to fibrin clot. Ancrod-induced fibrin formation could result in cerebral microvascular occlusion and may explain the suboptimal clinical effects of ancrod in human stroke trials.

Comparison of plasmin with recombinant tissue-type plasminogen activator in lysis of cerebral thromboemboli retrieved from patients with acute ischemic stroke.

BACKGROUND AND PURPOSE: Plasmin is a direct-acting thrombolytic with a better safety profile than recombinant tissue-type plasminogen activator (rtPA) in animal models. With the application of retrieval devices for managing acute ischemic stroke, extracted thromboemboli are available for ex vivo examination. We ask whether such thrombi are amenable to plasmin thrombolysis and whether such activity is different with rtPA. METHODS: Thromboembolic fragments (total 29) were retrieved from the intracranial carotid artery system of 15 patients with acute ischemic stroke and randomly assigned to ex vivo thrombolysis with plasmin or rtPA. After an initial 2-hour exposure, residual material was exposed to the other agent for an additional 2 hours. Thrombolysis was quantified by change in thrombus area and released d-dimer. RESULTS: Plasmin induced significant ex vivo thrombolysis of cerebral arterial thromboemboli, decreasing area by 45.9% ± 29.4% and 69.2% ± 52.5% and inducing median D-dimer release of 108,180 µg/L (range, 16,780 to 668,050 µg/L) and 16,905 µg/L (range, 240 to 403 085 µg/L) during the first and second 2-hour incubation periods, respectively. These changes were not different from those obtained with rtPA, which decreased area by 34.7% ± 57.8% (P=0.63) and by 68.4% ± 26.9% (P=0.97) and induced median D-dimer release of 151,990 µg/L (range, 9870 to 338,350 µg/L; P=0.51) and 34,520 µg/L (range 3794 to 325,400 µg/L; P=0.19) during the first and second 2-hour incubations. CONCLUSIONS: Retrieved human cerebral thromboemboli were amenable to ex vivo lysis by plasmin, the rate and degree of which was not different than that achieved with rtPA.

CT and MRI early vessel signs reflect clot composition in acute stroke.

BACKGROUND AND PURPOSE: The purpose of this study was to provide the first correlative study of the hyperdense middle cerebral artery sign (HMCAS) and gradient-echo MRI blooming artifact (BA) with pathology of retrieved thrombi in acute ischemic stroke. METHODS: Noncontrast CT and gradient-echo MRI studies before mechanical thrombectomy in 50 consecutive cases of acute middle cerebral artery ischemic stroke were reviewed blinded to clinical and pathology data. Occlusions retrieved by thrombectomy underwent histopathologic analysis, including automated quantitative and qualitative rating of proportion composed of red blood cells (RBCs), white blood cells, and fibrin on microscopy of sectioned thrombi. RESULTS: Among 50 patients, mean age was 66 years and 48% were female. Mean (SD) proportion was 61% (±21) fibrin, 34% (±21) RBCs, and 4% (±2) white blood cells. Of retrieved clots, 22 (44%) were fibrin-dominant, 13 (26%) RBC-dominant, and 15 (30%) mixed. HMCAS was identified in 10 of 20 middle cerebral artery stroke cases with CT with mean Hounsfield Unit density of 61 (±8 SD). BA occurred in 17 of 32 with gradient-echo MRI. HMCAS was more commonly seen with RBC-dominant and mixed than fibrin-dominant clots (100% versus 67% versus 20%, P=0.016). Mean percent RBC composition was higher in clots associated with HMCAS (47% versus 22%, P=0.016). BA was more common in RBC-dominant and mixed clots compared with fibrin-dominant clots (100% versus 63% versus 25%, P=0.002). Mean percent RBC was greater with BA (42% versus 23%, P=0.011). CONCLUSIONS: CT HMCAS and gradient-echo MRI BA reflect pathology of occlusive thrombus. RBC content determines appearance of HMCAS and BA, whereas absence of HMCAS or BA may indicate fibrin-predominant occlusive thrombi.

Haemostatic safety of a unique recombinant plasmin molecule lacking kringles 2-5.

We previously demonstrated a significant margin of haemostatic safety for full-length plasmin in comparison with tissue plasminogen activator (t-PA). We now report studies that compare haemostatic safety of full-length plasmin with a novel recombinant plasmin derivative, (Δ K2-5) plasmin, consisting of kringle 1 linked to the serine protease domain of plasmin. Agent was administered intravenously in a randomised, blinded manner in a rabbit model of fibrinolytic haemorrhage. A dose-related decrease in α2-antiplasmin, factor VIII, and fibrinogen followed administration of 1.8, 2.7, 3.7 and 4.6 mg/kg of (Δ K2-5) plasmin, with nadir fibrinogen concentrations of 65%, 40%, 30%, and 0% of initial levels, respectively. Mean primary bleeding time was undisturbed at 1.8 mg/kg (2.2 ± 0.7 minutes), minimally prolonged at 2.7 or 3.7 mg/kg (5 ± 2.9 and 4.4 ± 2.2 minutes), and prolonged at the purposefully toxic 4.6 mg/kg dose (12.8 ± 18.8 minutes). Equimolar amounts of (Δ K2-5) plasmin and full-length plasmin had equal in vitro clot lysis efficacy, but in the bleeding model, (Δ K2-5) plasmin showed better haemostatic competency than full-length plasmin. This safety advantage may be explained by higher residual amounts of plasma fibrinogen in animals given (Δ K2-5) plasmin rather than full-length plasmin. We demonstrate that a unique recombinant plasmin mutant, (Δ K2-5) plasmin, possesses an advantage in hemostatic safety over an equimolar amount of full-length plasmin.

Thrombolysis with plasmin: implications for stroke treatment.

Plasmin is a direct-acting thrombolytic agent with a striking hemostatic safety advantage over plasminogen activators in animal models of thrombolysis and bleeding. In contradistinction to plasminogen activators, which risk bleeding at any effective thrombolytic dose, plasmin is tolerated without bleeding at several-fold higher amounts than those needed for thrombolysis. Plasmin has been safe in a current trial in patients with peripheral arterial or graft occlusion, and efforts are now directed toward therapy of stroke caused by cerebral artery occlusion. A rabbit (4 kg body weight) model of 2-hour, thrombin-induced middle cerebral artery occlusion using angiographic documentation of vascular patency and recanalization was used to perform a dose-ranging study of plasmin, delivered by catheter over a median duration of 10 minutes. Plasmin induced early recanalization in all animals (3 per group) within 10 minutes after discontinuation of 3, 2, or 1 mg of agent infusion. Control saline infusion failed to induce recanalization in 3 of 3 subjects. Plasmin rapidly induces middle cerebral artery recanalization, as determined in an angiogram-based animal model of arterial occlusion. Based on these data and other information, a phase I/IIa clinical trial of plasmin in human middle cerebral artery ischemic stroke has been initiated.

Thrombolytic therapy for deep vein thrombosis: potential application of plasmin.

Plasmin is the prototype of a distinct class of "direct-acting" fibrinolytic agents, with biochemical and physiological attributes that are favorable for catheter-delivered thrombolytic therapy. Our studies indicate that plasmin is superior to plasminogen activators for hemostatic safety and thrombolytic efficacy in experimental models, and that plasmin has potential to avoid the bleeding risk that accompanies therapy of deep vein thrombosis with currently-used thrombolytic agents.

Pre-clinical studies of plasmin: superior benefit-to-risk ratio of plasmin compared to tissue plasminogen activator.

Currently-used thrombolytic agents are plasminogen activators (PA). While effective for treating thrombotic disease, PA use is associated with unavoidable hemorrhagic complications in susceptible individuals. Thus, there is an urgent need for new agents or approaches that provide greater hemostatic safety without sacrificing thrombolytic efficacy. Evidence now strongly indicates that 'direct-acting' thrombolytics, which do not require conversion of the precursor plasminogen to the active form plasmin, offer such a potential. The biochemical properties of plasmin provide a theoretical foundation for safe and effective therapy, based on thrombolytic efficacy upon local catheter delivery of agent and neutralization of circulating agent by its inhibitor (alpha-2 antiplasmin) to prevent bleeding complications. In vitro and animal studies support these predictions. In comparison with tissue-plasminogen activator (t-PA), plasmin shows a distinct benefit-to-risk advantage. First, plasmin is equally effective as t-PA in thrombolysis and may be superior for treating thrombi in totally-occluded vessels. Second, whereas t-PA causes bleeding from vascular trauma sites in animals when infused at dosages used for thrombolysis (0.5-1 mg/kg), plasmin exhibits safety at therapeutic dosages. In fact, plasmin can be used at several fold higher concentrations than is needed for thrombolysis, thereby providing a significant safety margin that is not attainable for t-PA or other PAs. Phase I trials with plasmin in hemodialysis graft occlusion and peripheral arterial occlusion have thus far confirmed the hemostatic safety of plasmin.

Middle cerebral artery occlusion in the rabbit using selective angiography: application for assessment of thrombolysis.

BACKGROUND AND PURPOSE: An animal model of selective middle cerebral artery (MCA) occlusion is needed for evaluation of intra-arterial (IA) delivery of thrombolytic agents. We describe a technique for MCA thrombo-occlusion in the rabbit with real-time angiographic documentation of occlusion and thrombolytic recanalization. METHODS: After femoral artery cutdown, a microcatheter was advanced from the internal carotid artery to the MCA. MCA occlusion was achieved by IA thrombin and reperfusion by IA plasmin. RESULTS: The terminal internal carotid artery was successfully catheterized in 12 of 13 animals. Stable (2-hour) MCA occlusion was induced and verified angiographically in all 12 animals; 2 animals also had distal internal carotid artery thrombus. Recanalization was achieved rapidly after IA plasmin in 3 of 3 animals. CONCLUSIONS: We describe a new animal model of selective MCA occlusion documented by real-time angiography and used to demonstrate recanalization with IA plasmin.

Analysis of thrombi retrieved from cerebral arteries of patients with acute ischemic stroke.

BACKGROUND AND PURPOSE: Information regarding the histological structure of thromboemboli that cause acute stroke provides insight into pathogenesis and clinical management. METHODS: This report describes the histological analysis of thromboemboli retrieved by endovascular mechanical extraction from the middle cerebral artery (MCA) and intracranial carotid artery (ICA) of 25 patients with acute ischemic stroke. RESULTS: The large majority (75%) of thromboemboli shared architectural features of random fibrin:platelet deposits interspersed with linear collections of nucleated cells (monocytes and neutrophils) and confined erythrocyte-rich regions. This histology was prevalent with both cardioembolic and atherosclerotic sources of embolism. "Red" clots composed uniquely of erythrocytes were uncommon and observed only with incomplete extractions, and cholesterol crystals were notably absent. The histology of thromboemboli that could not be retrieved from 29 concurrent patients may be different. No thrombus >3 mm wide caused stroke limited to the MCA, and no thrombus >5 mm wide was removed from the ICA. A mycotic embolus was successfully removed in 1 case, and a small atheroma and attached intima were removed without clinical consequence from another. CONCLUSIONS: Thromboemboli retrieved from the MCA or intracranial ICA of patients with acute ischemic stroke have similar histological components, whether derived from cardiac or arterial sources. Embolus size determines ultimate destination, those >5 mm wide likely bypassing the cerebral vessels entirely. The fibrin:platelet pattern that dominates thromboembolic structure provides a foundation for both antiplatelet and anticoagulant treatment strategies in stroke prevention.