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.

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.

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.

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.

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.

Locally delivered plasmin: why should it be superior to plasminogen activators for direct thrombolysis?

With advances in the field of thrombolytic therapy, whereby clots are routinely treated locally via a catheter, traditional systemic thrombolytics such as plasminogen activators might not be the best drugs for this task. Plasmin represents a new class of thrombolytic agents that exhibit direct fibrinolytic activity, without the need for either plasminogen or a plasminogen activator. In contrast to plasminogen activators, this independence from plasminogen allows plasmin to efficiently dissolve long, retracted blood clots that are inherently deficient in plasminogen. Preclinical safety studies in rabbits demonstrate that plasmin, in contrast to tissue-type plasminogen activator, does not cause re-bleeding from preformed hemostatic plugs. These results predict that plasmin will prove to be both superior to, and safer than, plasminogen activators in the dissolution of long, retracted blood clots in humans.

Quantitative assessment of thrombus burden predicts the outcome of treatment for venous thrombosis: a systematic review.

PURPOSE: Clot-burden change in patients receiving anticoagulant therapy, by predicting subsequent recurrent venous thromboembolism, may provide a clinically relevant surrogate endpoint of prognostic importance. The validity of this objective measure is yet to be established. METHODS: A PubMed search was performed to retrieve articles published up to December 2003. We identified 11 randomized trials reported from 1990 to 2003 that met our study identification and selection criteria. Anticoagulant therapy subsequently approved by regulatory affairs was assessed by clot-burden change and the validated outcome measure, long-term venous thromboembolism. Two additional randomized trials, partly meeting the inclusion criteria, were included in the sensitivity analysis. RESULTS: Individual studies suggested a predictive relationship between clot-burden change and likelihood of recurrent venous thromboembolism irrespective of the particular anticoagulant. The summary treatment effects strongly favored the therapy under evaluation and were in harmony for improved clot-burden (relative risk 0.82; 95% CI, 0.76-0.88; P <0.001) and for recurrent venous thromboembolism (relative risk 0.56; 95% CI, 0.42-0.76; P <0.001). The aggregate data show a striking predictive correlation for clot-burden change and subsequent recurrent venous thromboembolism using meta-regression analysis; (correlation = 0.81, P = 0.005) validating quantitative clot-burden assessment. CONCLUSION: Clot-burden change predicts long-term outcome, providing clinically relevant, patient-specific prognostic findings that may guide duration of anticoagulant therapy as well as provide a valid surrogate endpoint for clinical trials of innovative antithrombotic therapy, allowing more efficient trials exposing far fewer patients to the hazards of ineffective therapy than is required for outcome studies. Noninvasive assessment (duplex ultrasonography) of clot-burden change is currently being deployed for use in clinical trials.

Relationship between intermittent claudication, inflammation, thrombosis, and recurrent cardiac events among survivors of myocardial infarction.

BACKGROUND: Among coronary disease patients, concomitant peripheral arterial disease is a potent risk factor for future cardiac events and mortality. We sought to determine clinical and biochemical markers that might better elucidate the relationship between coronary and peripheral arterial disease. METHODS: Two months after an index myocardial infarction, 1045 patients provided detailed medical histories and underwent blood testing for selected hemostatic, lipid, and inflammatory markers. Patients were then followed up prospectively for a mean of 26 months. RESULTS: Compared with individuals without intermittent claudication (n = 966), those with claudication (n = 78) (information was unavailable for 1 individual) were significantly older and demonstrated an increased frequency of diabetes mellitus, tobacco use, prior cardiac and cerebrovascular events, and depressed left ventricular function. Individuals with claudication were less likely to receive beta-blocker therapy after the index infarction. Individuals with claudication had evidence of enhanced procoagulant and proinflammatory states manifested by relative elevations in plasma fibrinogen, D-dimer, C-reactive protein, and serum amyloid A concentrations. During follow-up, the presence of claudication was associated with an independent 2-fold increase in the combined end point of death or nonfatal cardiac event (38.5% vs 17.8%, P =.001) and a 5-fold increase in cardiac mortality (19.2% vs 3.6%, P =.001). Patients with intermittent claudication who were not treated with beta-blockers had a significant 3-fold mortality excess relative to those receiving beta-blockers. CONCLUSIONS: Following myocardial infarction, the added presence of intermittent claudication is associated with heightened procoagulant and proinflammatory states and an underuse of beta-blocker therapy and is a strong independent predictor of recurrent cardiovascular events.

Towards safer thrombolytic therapy.

Plasminogen activators (PA) are unique agents that are currently applied as thrombolytic therapy to achieve rapid vascular reperfusion. Regimens of PA plus anticoagulants and antiplatelet drugs have attained a high degree of sophistication and predictable rates of positive clinical outcomes for acute myocardial infarction (MI), ischemic stroke, pulmonary embolism (PE), deep vein thrombosis (DVT), and thrombosed catheters. Included in the repertoire are newly approved mutants of tissue plasminogen activator (TPA), which have biochemical advantages that allow for bolus administration. Yet, despite tremendous effort devoted to enormous trials to establish the clinical efficacy of these agents in acute MI, mortality results are not superior to those with native TPA or streptokinase (SK). Furthermore, all PAs have the potential for hemorrhagic complication, most critically intracranial hemorrhage (ICH), occurring in 0.9% of patients treated with native or mutant TPA. It is possible that a limit of clinical effectiveness has been reached, beyond which more potent PAs do not achieve greater benefit without a serious increase in risk of bleeding. A breakthrough is possible, however, if the risk of ICH could be avoided. One solution is the application of the direct-acting thrombolytic enzyme, plasmin. While intravenous plasmin is not effective when administered systemically, regional infusion to a thrombus induces local thrombolysis. Unlike the PAs, plasmin treatment should not cause hemorrhage from vascular trauma sites, as it is neutralized by antiplasmin in the blood. Animal studies are fully consistent with this approach, which offers potential for achieving a truly regional thrombolytic treatment.

Metabolic syndrome best defines the multivariate distribution of blood variables in postinfarction patients.

The hypothesis was tested that metabolic syndrome (MS) plays a leading role in approximating the multivariate distribution of thrombogenic and metabolic blood variables in a population of postinfarction patients. The multivariate statistical technique of factor analysis was used to determine blood variable clustering 2 months after myocardial infarction. Five clusters resulted in two separate independent factors, dyslipidemia and metabolic, reflecting MS and the remaining interpreted as cholesterol-lipoprotein, vascular-inflammatory, and coagulation. All five factors accounted for 55% of total variance with MS-associated factors accounting for 20% and individual factor contributions as follows: 11.6, 8.6, 12.9, 11.9, and 9.6%, respectively. There were no interactions of metabolic variables with thrombogenic variables or CRP in any factor. Results of subgroup analysis in males and females and in patients on and not on statins were all similar to the total group. We conclude there is no interaction of variables of MS or cholesterol-lipoprotein factors with those of thrombogenic factors. This independence yields the potential for use of factors in evaluating CVD risk. Further, the importance of MS in this group of postinfarction patients is emphasized, as the largest contribution to total variance was from MS factors, meaning that these variables best approximate the original multivariate distribution.

Foundation and sites of action of antithrombotic agents.

Thromboembolic disorders are a major cause of morbidity and mortality. As knowledge of the complex interactions between the vessel wall, platelets and coagulation and fibrinolytic enzyme systems increases, new avenues for more effective and safer therapies become evident. In this review, we discuss mechanisms of hemostasis in relation to antithrombotic agents and results of clinical trials using these drugs.

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.

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.