Approach to the treatment, characterization and diagnosis of an acquired auto-antibody directed against factors prothrombin, factor X and factor IX: a case report and review of the literature.

Bleeding disorders secondary to acquired non-inhibitory antibodies directed against vitamin K-dependent coagulation proteins are rare. In this report, the authors describe a patient with a low grade lymphoma who presented with a fatal acquired bleeding manifestation and abnormal hemostatic studies resulting from deficiencies in both prothrombin and factor X. Patient plasma samples were collected and studied for the presence of an acquired inhibitor. Levels of plasma coagulation proteins were measured using immunoassay. Patient anti-prothrombin immunoglobulin G was isolated and binding to prothrombin, prothrombin F1.2, factors IX and X was evaluated using immunoblots and competition immunoassay. Prolongation in the prothrombin time and activated partial thromboplastin time suggested a factor deficiency in the common pathway of coagulation. Functional and antigenic levels of both prothrombin and factor X were decreased. An IgG subtype-4 antibody was isolated from patient plasma using affinity chromatography on prothrombin-sepharose. This antibody was found to bind to a common metal-ion-dependent conformational epitope found on the γ-carboxyglutamic acid (Gla) domain of prothrombin, factor X and factor IX. This report represents the first description of an acquired bleeding disorder resulting from a unique cross-reactive auto-antibody against a common metal-ion-dependent antigenic structure on the Gla-domain of the vitamin K-dependent proteins.

The neurovascular unit in the setting of stroke.

Microvessels and neurons respond rapidly and simultaneously in focal regions of ischaemic injury in such a way as to suggest that the responses could be coordinated. The ability of neurons to modulate cerebral blood flow in regions of activation results from neurovascular coupling. But little is known about the microvessel-to-neuron direction of the relationship. The presence and participation of intervening glial cells implies the association of microvessels, glia, and neurons in a 'neurovascular unit'. The interdependent functions of the cellular and matrix components of this theoretical unit have not been rigorously explored, except under conditions of injury where, for the most part, only single components or tissue samples have been studied. Whereas maintenance or timely re-establishment of flow reduces tissue and neuron injury in both humans and animal models, protection of neuron function in humans has not prevented the evolution of injury despite the inherent mechanisms of neurovascular coupling. However, occlusion of flow to the brain rapidly identifies regions of neuron-vascular vulnerability within the vascular territory-at-risk. These coalesce to become the mature ischaemic lesion. The failure, so far, of clinical trials of neuron protectant agents to achieve detectable tissue salvage could be explained by the vulnerability (and lack of protection) of essential components of the 'unit'. This presentation summarizes evidence and thoughts on this topic. These support the need to understand component interactions within the neurovascular unit.

Inflammation and the neurovascular unit in the setting of focal cerebral ischemia.

Responses to focal cerebral ischemia by neurons and adjacent microvessels are rapid, simultaneous, and topographically related. Recent observations indicate the simultaneous appearance of proteases by components of nearby microvessels that are also expressed by neurons in the ischemic territory, implying that the events could be coordinated. The structural relationship of neurons to their microvascular supply, the direct functional participation of glial cells, and the observation of a highly ordered microvessel-neuron response to ischemia suggest that these elements are arranged in and behave in a unitary fashion, the neurovascular unit. Their roles as a unit in the stimulation of cellular inflammation and the generation of inflammatory mediators during focal cerebral ischemia have not been explored yet. However, components of the neurovascular unit both generate and respond to these influences under the conditions of ischemia. Here we briefly explore the potential inter-relationships of the components of the neurovascular unit with respect to their potential roles in ischemia-induced inflammatory responses.

Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation. METHODS: The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-microm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O). RESULTS: Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA-PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned. CONCLUSIONS: The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury.

Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Antiplatelet therapy in thrombotic thrombocytopenic purpura.

The presence of platelet-fibrin microthrombotic occlusions in the arterioles and capillaries of involved tissues of patients with TTP has suggested a role for platelet aggregation in this disorder. Inhibitors of platelet aggregation have been reported to produce resolution of thrombocytopenia and clinical improvement in many instances. Failure of such agents to produce a clinical effect has been attributed to inadequate dose-duration, severity and extent of end organ/vascular involvement, long-term or secondary effects of the etiologic principal leading to patient deterioration and/or demise. On the other hand, the parallel use of several treatment modalities that themselves may produce clinical effect confounds the interpretation that anti-platelet agents alone may have been responsible for clinical improvement. Nonetheless, complete remission has been reported in a number of TTP patients when the combination ASA/dipyridamole was used alone or together with plasmapheresis, splenectomy, and/or other antiplatelet agents. The evidence for a beneficial clinical effect would seem strongest for the use of this combination early in the course of the disease. More limited and less conclusive has been the experience with sulfinpyrazone, with ticlopidine, and with intravenous PGI2 infusions in TTP. Reports of clear-cut benefit with each of these agents have been rare. Finally, serial dextran infusions have apparently produced amelioration of the clinical syndrome in certain individuals. Assessment of benefit of dextran infusions from retrospective series has been limited by antecedent use of splenectomy. The use of red cell and plasma infusions during splenectomy has been argued to provide some benefit. However, it is likely that dextran can produce definite responses in certain patients. Unfortunately, therapeutic efficacy has been judged from such anecdotal reports and retrospective series. No prospective controlled trials of any of these approaches are available.

Hemorrhagic transformation and microvascular integrity during focal cerebral ischemia/reperfusion.

Hemorrhagic transformation after cerebral ischemia is a well known clinical concern. The frequency of intact basal lamina (BL), identified by laminin antigen, in hemorrhagic and nonhemorrhagic zones after middle cerebral artery occlusion (MCA:O) and 3-h MCA:O with reperfusion in adolescent male baboons was assessed. Parenchymal hemoglobin was not detected prior to 24-h reperfusion. A significant decrease in the density of laminin (BL) in hemorrhagic zones (6.2 +/- 2.4) compared with nonhemorrhagic ischemic zones (10.5 +/- 2.4) (p < 0.05) and nonischemic basal ganglia (17.0 +/- 2.7) (p < 0.01) was observed. Time-dependent changes in BL integrity appear linked to the extravasation of blood components.

Experimental antileukocyte interventions in cerebral ischemia.

White blood cells (WBCs) play vital roles in host defense. Recently, increasing interest has been directed toward the question of whether WBCs, particularly polymorphonuclear leukocytes, could also act as mediators of secondary brain damage in the setting of focal and global cerebral ischemia with and without reperfusion. Considerable insight into the importance of WBC-mediated tissue injury has been gained from studies employing antileukocyte interventions in experimental cerebral ischemia. The purpose of this article is to survey the different approaches taken to interfere with WBC inflammatory function. Emphasis is laid on a discussion of the efficacy of these interventions, their effects and side effects on cerebral and systemic parameters, and the power of evidence they provide for identification of WBCs as important factors in cerebral ischemia. The role of WBCs has been investigated in a great variety of global and focal cerebral ischemia models with and without reperfusion, leading to sometimes contradictory results. In the light of currently available data, it seems likely that WBCs contribute to secondary brain damage in the scenario of experimental transient focal cerebral ischemia, if the insult is not too severe.

Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94.

It has been shown recently that matrix metalloproteinases (MMPs) are elevated after cerebral ischemia. In the current study, we investigated the pathophysiologic role for MMP-9 (gelatinase B, EC.3.4.24.35) in a mouse model of permanent focal cerebral ischemia, using a combination of genetic and pharmacologic approaches. Zymography and Western blot analysis demonstrated that MMP-9 protein levels were rapidly up-regulated in brain after ischemic onset. Reverse transcription polymerase chain reaction showed increased transcription of MMP-9. There were no differences in systemic hemodynamic parameters and gross cerebrovascular anatomy between wild type mice and mutant mice with a targeted knockout of the MMP-9 gene. After induction of focal ischemia, similar reductions in cerebral blood flow were obtained. In the MMP-9 knockout mice, ischemic lesion volumes were significantly reduced compared with wild type littermates in male and female mice. In normal wild type mice, the broad spectrum MMP inhibitor BB-94 (batimastat) also significantly reduced ischemic lesion size. However, BB-94 had no detectable protective effect when administered to MMP-9 knockout mice subjected to focal cerebral ischemia. These data demonstrate that MMP-9 plays a deleterious role in the development of brain injury after focal ischemia.

Polymorphonuclear leukocyte behavior in a nonhuman primate focal ischemia model.

There is increasing interest in the role of polymorphonuclear (PMN) leukocytes in the evolution of focal cerebral infarction. Surgical preparation of focal cerebral ischemia models may alter leukocyte reactivity and thereby make interpretation of leukocyte function following ischemia/reperfusion difficult. The effects of surgical preparation and of experimental ischemia/reperfusion on granulocyte function have been examined prospectively in a baboon model. Twenty-six adolescent male baboons underwent surgical preparation, of which 21 underwent middle cerebral artery occlusion/reperfusion. Four additional animals served as nonsurgical controls. Peripheral venous blood specimens were taken for performing assays of leukocyte function at defined intervals before and after both the surgical preparation (i.e., the overall procedure for implantation of the middle cerebral artery occlusion device) and occlusion/reperfusion. A stress-related elevation in total leukocyte number was attributed mainly to an increase in the number of circulating PMN leukocytes. Values rose from 13.9 +/- 4.9 x 10(3) to 27.8 +/- 5.8 x 10(3)/microliters, (+/- SD; n = 21) for total leukocyte number, with p < 0.001, and from 4.3 +/- 2.1 x 10(3) to 15.9 +/- 4.7 x 10(3)/microliters (n = 21) for PMN leukocytes, with p < 0.001. Surgical preparation had no effect (p > or = 0.4) on the ability of PMN leukocytes, isolated 24 h after the implantation procedure, to display polarization, O2.- production, or beta-glucuronidase release when stimulated with human C5a. A moderate decrease in the chemotactic response to C5a resolved within the 7-day postsurgery (preocclusion) period. Three-hour middle cerebral artery occlusion and 1-h reperfusion resulted in a significant reduction in C5a-induced polarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia.

Both vascular endothelial growth factor (VEGF) and integrin alpha(v)beta3 play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin alpha(v)beta3 expression. However, it is unknown whether VEGF, like integrin alpha(v)beta3, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP+) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin alpha(v)beta3 were expressed by activated noncapillary (7.5- to 30.0-microm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin alpha(v)beta3+, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin alpha(v)beta3, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit alpha(v) messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin alpha(v)beta3 together.

Matrix metalloproteinases increase very early during experimental focal cerebral ischemia.

Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-microm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.

Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury.

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

The role of platelets in ischemic stroke.

Platelets have assumed a role in the development of focal cerebral ischemia by virtue of their participation in thromboemboli that may initiate stroke symptoms. Platelets are one component of the blood-vascular axis responsible for preventing hemorrhage. Activated platelets initiate hemostatic plug formation and provide a scaffolding for coagulation activation. Platelets are activated by a number of stimuli, such as exposure of the vascular subendothelium, fibrin deposition, and abnormal surfaces, e.g., atheromata. A number of observations, including the appearance of platelet thrombi on atheromata in situ, indicate that platelet physiology is relevant to stroke. In addition, certain antiplatelet agents (e.g., aspirin) significantly reduce the incidence of ischemic stroke after initial transient ischemic attacks. Aspirin, the combination of aspirin and dipyridamole, and ticlopidine have all been shown to be useful in reducing the frequency of secondary stroke events. Clopidogrel has been shown to reduce the frequency of secondary vascular ischemic events when stroke, myocardial infarction, and peripheral arterial disease are considered together. Unfortunately, all antithrombotic agents carry a potential risk for inducing symptomatic intracerebral hemorrhage during ischemic stroke. The mechanism by which this may happen with antiplatelet agents has not yet been determined. As in other areas of stroke treatment, it is the balance between efficacy in reduction of symptomatic thrombotic events and the risk for hemorrhage that will define benefit.

Clinical trials in acute stroke: why have they not been successful?

Modeling of focal cerebral ischemia seeks to understand mechanisms of injury and to test agents as potential stroke therapies. However, modeling has been singularly unpredictable in ischemic cerebrovascular disease for a number of reasons related to the incompletely understood pathophysiology of ischemic stroke and to the characteristics of models prepared to mimic the clinical condition. The development of models of focal cerebral ischemia must take into account known species differences and idiosyncrasies, underlying vascular disease processes, the nature of thrombotic processes, cellular reactivities, the presence of co-stimulation (e.g., inflammation), the characteristics of immunologicals and reporter molecules, the coincident use of other pharmacologic modifiers (e.g., anesthesia), and stress. These elements are also potential contributors to cerebral tissue injury and its assessment but may affect other species differentially. On the other hand, study design issues have been shown to be particularly relevant to limiting development of some agents for clinical stroke treatment. Experience from experimental and clinical work on vascular active approaches (e.g., plasminogen activators) suggests that active dialogue regarding the relationships between clinical outcomes and outcomes in appropriate animal models is necessary. Success appears more likely when the model more closely matches the known human pathophysiology and the interventions applied in the models are definitely characterized in that species. Rather than moving directly to interventional studies in humans, the use of several appropriate animal models is encouraged where those models exist. Where not, careful consideration of study design and the biology of the disorder is a prerequisite.

Intracranial hemorrhage after coronary thrombolysis with tissue plasminogen activator.

PURPOSE AND METHODS: We analyzed the clinical, laboratory, and radiologic data in nine patients who sustained an intracranial hemorrhage (ICH) after receiving intravenous recombinant tissue plasminogen activator (rt-PA) and heparin for treatment of acute myocardial infarction (MI). Our purpose was to delineate the clinical and radiologic features of the ICHs, as well as to determine their potential risk factors and mechanisms. RESULTS: Among 1,700 patients with an acute MI treated with an investigational two-chain rt-PA, duteplase (Burroughs Wellcome Co., Research Triangle Park, NC), nine (0.53%) developed symptomatic ICH. Neurologic symptoms occurred between 7 and 96 hours after onset of rt-PA therapy. All patients received heparin concomitantly for prevention of coronary reocclusion. The activated partial thromboplastin times (aPTTs) in five of eight (63%) patients at onset of ICH were excessively prolonged (greater than two times control); hypofibrinogenemia occurred in only one of five (20%) patients tested; and thrombocytopenia was present in only one of the nine (11%) patients. Fibrin degradation products (FDPs) were elevated in all five patients tested. Minor hemorrhage (not requiring transfusion) outside the central nervous system occurred in five of the nine patients with ICH. The ICHs were often of lobar location and of moderate to large size. They occurred at multiple sites in three patients, and were fatal in four instances (44%). CONCLUSIONS: The incidence of ICH in this series was low, and consistent with figures reported from studies with alteplase in patients with acute MI. The mechanisms of these hemorrhages remain unclear; while hypofibrinogenemia was not a uniform finding, excessive prolongation of the aPTT and elevated FDPs may have contributed to the occurrence of ICH in some patients. Still unidentified local cerebrovascular factors may play an additional role in causing ICH. In order to further clarify the mechanisms of ICH in the setting of thrombolytic therapy, prospective data collection on probable risk factors for ICH in patients with acute MI treated with rt-PA will be required.

Antithrombotic therapy of acute stroke: thrombolytic agents.

Following the initial report of the clinical use of plasminogen activators (PAs) in cerebrovascular thrombosis in 1958, interest in the efficacy of this approach accelerated only when the need for acute intervention in stroke was recognized. The use of PAs in acute ischemic stroke is based on the observation that approximately 80-90% of focal cerebral ischemic events presenting as strokes within 8 hr of symptom onset are due to atherothrombotic and embolic occlusions.

Antithrombotic treatments in acute ischemic stroke.

A therapeutic role for antiplatelet agents and anticoagulants within 6 hours of the onset of ischemic stroke symptoms has not been tested. With one exception, their use in early stroke (< 48 hours) did not produce a favorable outcome. The use of rt-PA in appropriate patients presenting with ischemic stroke within 3 hours of symptom onset has been accompanied by significant benefit, which has exceeded the risk of intracerebral hemorrhage in one trial. The recent group of clinical studies has provided evidence for factors which may contribute to hemorrhagic transformation. These studies also demonstrate the following: i) recanalization of carotid and vertebrobasilar artery territory occlusions is technically feasible within 3 to 6 hours of symptom onset, ii) the frequency of hemorrhage is increased in ischemic stroke patients receiving PAs, iii) the interval from symptom onset to treatment to achieve clinical improvement varies individually and contributes to hemorrhagic risk, and iv) the optimal PAs, their dose-rate, and delivery systems have not yet been defined in either the carotid or vertebrobasilar territory. Taken together, the NINDS trial, ECASS, and ECASS II indicate the enormous importance of patient selection to reduce the hemorrhagic risk which accompanies the use of PAs in stroke. However, it is currently not possible to separate benefit from hemorrhagic risk in a given patient based upon simple clinical criteria, although contributors to this risk have been identified. Clearly, attempts to reduce the risk of hemorrhage should contribute to the overall benefit of selected ischemic stroke patients treated with rt-PA and with other PAs. This experience may also provide a clinical basis for the prospective study of antiplatelet agents and anticoagulants in acute ischemic stroke.